Enzymatic regioselective polymerization for preparing linear polylysine or polyarginine

Abstract

The present invention relates to a method for preparing linear polylysine or polyarginine by chemoenzymatic bulk polymerization of lysine esters or arginine esters, respectively. The invention also relates to a method for controlling the regioselectivity of polylysine or polyarginine in the chemoenzymatic bulk polymerization of lysine esters or arginine esters.

Description

Technical Field

[0001] This invention relates to a method for preparing linear polylysine or polyarginine via chemiluminescent bulk polymerization of lysine esters or arginine esters, respectively. The invention also relates to a method for controlling the regioselectivity of polylysine or polyarginine during the chemiluminescent bulk polymerization of lysine esters or arginine esters. Background Technology

[0002] Over the past few decades, polylysine and its derivatives have been studied in numerous fields, such as as preservatives in food and personal care products, as surfactants and moisturizers in personal care and household products, as a nutrient source in nutritional supplements, and as bioactive ingredients in cosmetics or dermatological products. Among the various methods used to synthesize polylysine, enzymatic polymerization has attracted increasing attention due to its advantages in milder reaction conditions, fewer side reactions, higher selectivity, environmental friendliness, and simpler operation without the need for cumbersome protection-deprotection processes.

[0003] Enzyme-catalyzed polymerization for the preparation of polylysine is typically carried out in single-phase or multiphase systems using proteases in the presence of water. It is believed that the synthesis of water-insoluble amino acid oligomers catalyzed by proteases in aqueous media is precipitation-driven. The synthesis of water-soluble oligomers of amino acids such as lysine can only be controlled in mixed-phase systems, where the equilibrium shifts in favor of peptide synthesis due to enhanced partitioning of the peptide in the organic phase.

[0004] US 2006 / 0252134 A1 describes a method for the chemically enzymatic polymerization of hydroxycarboxylic acids and amino acids. In this method, oligolysine is prepared from commercially available lysine ethyl ester dihydrochloride as a substrate for papain. Multiphase systems are described: a two-phase system comprising an aqueous suspension of papain and toluene containing the substrate; a reverse micelle system comprising an aqueous solution of papain and a reverse micelle solution of the substrate in isooctane; and a three-phase system comprising an aqueous suspension of papain and the substrate in another two phases of octane and decafluoropentane. The obtained oligolysine has a degree of polymerization between 2 and 9, with a peak maximum of 4. This implies that only the α-group is reactive, at least in the case of lysine.

[0005] FR 2708938 A1 describes a chemoenzymatic method for preparing L-lysine oligomers using lysine ethyl ester dihydrochloride and trypsin. Polymerization is carried out in a water-isopropanol mixture until two or more phases are formed. The degree of polymerization ranges from 2 to 8, with a peak maximum of 3.3; however, the regioselectivity of the products is not discussed.

[0006] CN 112226469 A describes a chemical enzymatic method for preparing L-lysine oligomers using commercially available lysine methyl ester dihydrochloride and trypsin, papain, or alkaline protease in a solvent mixture of an aqueous buffer and dimethyl sulfoxide at pH 6.5 to 8. The degree of polymerization or regioselectivity of the obtained oligopeptides is not discussed.

[0007] CN 110669810 A describes a chemical enzymatic method for preparing L-lysine oligomers using commercially available lysine methyl ester dihydrochloride and cross-linked bromelain, papain, or neutral protease in a solvent mixture consisting of an aqueous buffer solution at pH 5.5 to 8 and tert-butanol. The degree of polymerization or regioselectivity of the obtained oligopeptides is not discussed.

[0008] CN 112301081 A describes a chemical enzymatic method for preparing L-lysine oligomers using commercially available lysine methyl ester dihydrochloride and trypsin in an aqueous buffer at pH 7 to 8.5. The degree of polymerization or regioselectivity of the obtained oligopeptides is not discussed.

[0009] Srinivasan et al. reported the preparation of papain-catalyzed oligopeptides in *Papain Catalyzed Oligomerization in Monophasic Aqueous Organic Media - Synthesis and Characterization of Neutral and Polar Amino Acid Oligomers*, *Enz. Eng* [Enzyme Engineering] 2017, 6:161. Specifically, oligolysine was obtained in a monophasic water-acetonitrile system using commercially available lysine ethyl ester dihydrochloride. The degree of polymerization ranged from 2 to 10, with a peak maximum of 5; however, regioselectivity was not discussed.

[0010] Aso et al. reported the preparation of oligolysine in Trypsin-catalyzed Oligomerization of L-Lysine Esters, Bioscience, Biotechnology, 56:5, 755-758 (1992). The preparation was carried out in aqueous solution at pH 10 in the presence of NaCl using commercially available ethyl, n-butyl, or n-hexyl lysine dihydrochloride. The degree of polymerization ranged between 2 and 8, with a peak maximum of 4. The interaction between the α-amino group of lysine and the enzyme was also discussed.

[0011] Puigserver et al. reported the preparation of papain-catalyzed oligo-lysine in *Papain-catalyzed Polymerization of Amino Acids in Low-Water Organic Solvents*, *Biotechnology Letters*, Vol. 13, No. 13, pp. 161-166 (1991). This preparation utilizes lysine methyl or ethyl dihydrochloride prepared from lysine and thionyl chloride in the corresponding alcohol solution, and polyethylene glycol-modified papain. Polymerization is carried out in acetonitrile containing a small amount of water. The degree of polymerization of lysine ranges from 2 to 12, with peak maxima between 4 and 6; however, regioselectivity is not discussed.

[0012] Gross et al. reported the preparation of protease-catalyzed oligo-lysine in Aqueous Solution in *Protease-Catalyzed Oligomerization of Commercially Available L-Lysine Ethyl Ester in Aqueous Solution*, *ACS Catalysis*, 2011, 1, 9, 1022-1034. This preparation utilizes commercially available L-lysine ethyl ester dihydrochloride with bromelain, trypsin, chymotrypsin, or papain as proteases in an aqueous buffer. The degree of polymerization can reach up to 12, with a peak maximum of 3.6. Oligo-α-lysine was obtained with all proteases used.

[0013] To date, the preparation of polylysine via enzymatic bulk polymerization has never been reported. Only the enzymatic polymerization of amino acids with aspartic esters as substrates has been reported in bulk. For example, Matsumura et al. reported the bulk polymerization of diethyl aspartate using a commercially available basophilic protease in *Enzyme-catalyzed Polymerization of L-Aspartate*, *Macromol. Rapid Commun.* 20, 7-11 (1999). Poly-α-aspartate with up to 88% α-linkage and up to 23 repeating units was obtained. The polymer was saponified to yield sodium polyaspartate. Zhang et al. reported the bulk polymerization of diethyl aspartate using the immobilized lipase CAL-B in *Solvent-free Lipase-catalyzed Synthesis: Unique Properties of Enantiopure D- and L-Polyaspartates and Their Complexation*, *Biomacromolecules*, 2016, 17, 362-370, with up to 98% β-linking and an average of 63 repeating units. However, Zhang et al.'s analytical data on the polyaspartate linkages were challenged in Gross et al.'s subsequent publication, *CAL-B Catalyzed Regioselective Bulk Polymerization of L-Aspartic Acid Diethyl Ester to α-Linked Polypeptides*. Gross et al. demonstrated that, using immobilized CAL-B under the same bulk conditions as reported by Zhang et al., the polymerization of diethyl aspartate should result in a 94% α-linked polyaspartate with an average of 50 repeating units.

[0014] The synthesis of linear polylysine with high regioselectivity of α-polylysine or ε-polylysine via chemoenzymatic bulk polymerization of lysine esters, or with high regioselectivity of α-polyarginine via chemoenzymatic bulk polymerization of arginine esters, is challenging. Summary of the Invention

[0015] The object of this invention is to provide α- or ε-polylysine or α-polyarginine with desired regioselectivity more efficiently.

[0016] The inventors have discovered that this objective can be achieved by preparing linear polylysine or polyarginine through the chemical enzymatic polymerization of lysine ester or arginine ester in bulk, respectively.

[0017] Therefore, in a first aspect, the present invention relates to a method for preparing polylysine or polyarginine, the method comprising chemically enzymatically polymerizing a lysine ester in bulk in the presence of an immobilized protease to regioselectively provide α-polylysine, or chemically enzymatically polymerizing a lysine ester in the presence of an immobilized lipase to regioselectively provide ε-polylysine, or chemically enzymatically polymerizing an arginine ester in bulk in the presence of an immobilized protease to regioselectively provide α-polyarginine.

[0018] In some embodiments according to the first aspect, the present invention relates to a method for preparing polylysine, the method comprising:

[0019] - Make lysine C 1-6 Desalting alkyl or aryl ester salts to provide lysine C 1-6 -alkyl or aryl esters, and

[0020] - Make lysine C 1-6 -Alkyl or aryl esters are chemically enzymatically polymerized in the bulk in the presence of immobilized proteases to regioselectively provide α-polylysine, or chemically enzymatically polymerized in the presence of immobilized lipases to regioselectively provide ε-polylysine.

[0021] In some other embodiments according to the first aspect, the present invention relates to a method for preparing polyarginine, the method comprising:

[0022] - Make arginine C 1-6 -Desalting of alkyl or aryl ester salts to provide arginine C 1-6 -alkyl or aryl esters, and

[0023] - Make arginine C 1-6 -Alkyl or aryl esters undergo chemical enzymatic polymerization in the bulk in the presence of immobilized proteases to regioselectively provide α-polyarginine.

[0024] In some preferred embodiments according to the first aspect, the present invention relates to a method for preparing polylysine, the method comprising:

[0025] - Make lysine C 1-6 Desalting of inorganic acid salts of alkyl or aryl esters to provide lysine C 1-6 -alkyl or aryl esters, and

[0026] - Make lysine C 1-6 -Alkyl or aryl esters are chemically enzymatically polymerized in bulk in the presence of immobilized proteases selected from serine proteases (such as trypsin and subtilisin) or cysteine ​​proteases (such as papain) to regioselectively provide α-polylysine.

[0027] In some preferred embodiments according to the first aspect, the present invention relates to a method for preparing polylysine, the method comprising:

[0028] - Make lysine C 1-6 Desalting of inorganic acid salts of alkyl or aryl esters to provide lysine C 1-6 -alkyl or aryl esters, and

[0029] - Make lysine C 1-6 -Alkyl or aryl esters undergo chemical enzymatic polymerization in bulk in the presence of immobilized lipases, preferably immobilized lipases selected from the group consisting of: Candida Antarctica lipase B, Aspergillus Niger lipase, Burkholderia Cepacia (Pseudomonas Cepacia) lipase, and Rhizopus Oryzae lipase, to regioselectively provide ε-polylysine.

[0030] In some other preferred embodiments according to the first aspect, the present invention relates to a method for preparing polyarginine, the method comprising:

[0031] - Make arginine C 1-6 Desalting of inorganic acid salts of alkyl or aryl esters to provide arginine C 1-6 -alkyl or aryl esters, and

[0032] - Make arginine C 1-6 -Alkyl or aryl esters are chemically enzymatically polymerized in bulk in the presence of immobilized proteases selected from serine proteases (such as trypsin and subtilisin) or cysteine ​​proteases (such as papain) to regioselectively provide α-polyarginine.

[0033] In a second aspect, the present invention relates to a method for controlling the regioselectivity of linear polylysine or linear polyarginine in the chemical enzymatic polymerization of lysine esters or arginine esters in bulk, wherein an immobilized protease is used to regioselectively provide α-polylysine or α-polyarginine, or an immobilized lipase is used to regioselectively provide ε-polylysine.

[0034] In some embodiments according to the second aspect, the present invention relates to a method for separately processing lysine C 1-6 -alkyl or aryl esters or arginine C 1-6 A method for controlling the regioselectivity of linear polylysine or linear polyarginine in the chemical enzymatic polymerization of alkyl or aryl esters in bulk, wherein an immobilized protease selected from the group consisting of serine proteases (such as trypsin and subtilisin) and cysteine ​​proteases (such as papain) is used to regioselectively provide α-polylysine or α-polyarginine.

[0035] In some other embodiments according to the second aspect, the present invention relates to a method for [using] lysine C 1-6 A method for controlling the regioselectivity of linear polylysine in the chemical enzymatic polymerization of alkyl or aryl esters in bulk, wherein immobilized lipases, preferably selected from the group consisting of Candida antarcticis lipase B, Aspergillus niger lipase, Aspergillus niger lipase, Burkholderia cepacia lipase and Rhizopus oryzae lipase, are used to regioselectively provide ε-polylysine.

[0036] In a third aspect, the present invention relates to a method for preparing polylysine or polyarginine, the method comprising chemically enzymatically polymerizing lysine esters in bulk in the presence of an immobilized protease to regioselectively provide α-polylysine, or chemically enzymatically polymerizing lysine esters in the presence of an immobilized lipase to regioselectively provide ε-polylysine, or chemically enzymatically polymerizing arginine esters in bulk in the presence of an immobilized protease to regioselectively provide α-polyarginine.

[0037] In a fourth aspect, the present invention relates to a method for controlling the regioselectivity of linear polylysine or linear polyarginine in the chemical enzymatic polymerization of lysine esters or arginine esters in bulk, wherein an immobilized protease is used to regioselectively provide α-polylysine or α-polyarginine, or an immobilized lipase is used to regioselectively provide ε-polylysine.

[0038] The inventors have unexpectedly discovered that polylysine with high α-regioselectivity or ε-regioselectivity and polyarginine with high α-regioselectivity can be provided by the method according to the first aspect or the method according to the second aspect. In particular, polylysine with ε-regioselectivity can be obtained, which is unknown in the field of preparing polylysine via chemical enzymatic polymerization. Detailed Implementation

[0039] The singular forms “a / an” and “the” include plural indicators unless the context clearly specifies otherwise. The terms “comprise(s) / comprising” are interchangeable with “contain(s) / containing” and should be interpreted in a non-restrictive, open-ended manner. That is, for example, additional components or elements may exist. Expressions such as “consist(s) of / consisting of” or cognates may be included in “comprise(s) / comprising” or cognates. The terms “include(s) / including” should be interpreted in a non-restrictive, open-ended manner.

[0040] In this document, the term "regioselectivity" refers to a reaction that is highly favorable to a single major product. In the context of polylysine, "regioselectivity" is intended to refer to the percentage of α-polylysine or ε-polylysine in polylysine, such as in the chemical enzymatic polymerization of lysine esters. In the context of polyarginine, "regioselectivity" is intended to refer to the percentage of α-polylysine in polyarginine, such as in the chemical enzymatic polymerization of arginine esters.

[0041] In this article, the phrase “…chemical enzymatic polymerization in bulk” can also be referred to as “chemical enzymatic bulk polymerization”.

[0042] In this paper, the terms "lysine ester" and "arginine ester" refer to the corresponding free base forms of lysine ester and arginine ester.

[0043] In a first aspect, the present invention provides a method for preparing polylysine or polyarginine, the method comprising chemically enzymatically polymerizing a lysine ester in bulk in the presence of an immobilized protease to regioselectively provide α-polylysine, or chemically enzymatically polymerizing a lysine ester in the presence of an immobilized lipase to regioselectively provide ε-polylysine, or chemically enzymatically polymerizing an arginine ester in bulk in the presence of an immobilized protease to regioselectively provide α-polyarginine.

[0044] The present invention further provides a method for preparing polylysine or polyarginine, the method comprising chemically enzymatically polymerizing lysine esters in bulk in the presence of immobilized or non-immobilized proteases to regioselectively provide α-polylysine, or chemically enzymatically polymerizing lysine esters in the presence of immobilized or non-immobilized lipases to regioselectively provide ε-polylysine, or chemically enzymatically polymerizing arginine esters in bulk in the presence of immobilized or non-immobilized proteases to regioselectively provide α-polyarginine.

[0045] Suitable proteases for catalyzing peptide formation reactions with α-polylysine regioselectivity or α-polyarginine regioselectivity may include, but are not limited to, serine proteases (e.g., trypsin, α-chymotrypsin, elastase, carboxypeptidase, and subtilisin), cysteine ​​proteases (e.g., papain, fig protease, bromelain, analain, calpain, caspase, papain, cathepsin, clostridium protease, and actinidin), and metalloproteinases (e.g., thermophilic proteases, collagenases, Bacillus lysin). Bacillolysin, dispersase, vibriolysin, pseudolysin, matrix lysin, and various bacterial-derived neutral metalloproteinases), aspartic proteases (e.g., pepsin, penicillin pepsin, rennet, cathepsin, renin, β-secretase, Plasmodium plasma protease, and retroviral proteases), hepatic esterases (e.g., porcine liver esterase), alkaline proteases, carbonic anhydrase, nonribosomal peptide synthases, thrombin, cardosin A or B, or streptomycin.

[0046] In some embodiments, the protease used to catalyze peptide formation reactions having α-polylysine regioselectivity or α-polyarginine regioselectivity is selected from the group consisting of serine proteases (such as trypsin, α-chymotrypsin, elastase, carboxypeptidase, and subtilisin) and cysteine ​​proteases (such as papain, figolytic enzyme, bromelain, annalin, calpain, caspase, papain, cathepsin, clostridium protease, and actinidin). Specifically, the protease is selected from the group consisting of trypsin, subtilisin, and papain.

[0047] The enzymatic activity of proteases, particularly trypsin, can be measured using an Nα-benzoyl-DL-arginine-p-nitroaniline (BAPNA) assay, as fully described in the Examples section below. The trypsin used in this invention has an activity of at least 0.05 U / g immobilized trypsin, preferably at least 0.1 U / g, and more preferably at least 0.3 U / g.

[0048] Suitable lipases for catalyzing peptide formation reactions with ε-polylysine regioselectivity may include, but are not limited to, lipases (EC3.1.1.3), such as lipases from Candida antarctica B, and lipases from species of the genera Alcaligenes, Aspergillus, Mucor, Penicillium, Geotricum, Rhizopus, Burkholderia, Candida, Pseudomonas, and Thermomyces.

[0049] In some embodiments, the lipase used to catalyze a peptide formation reaction having ε-polylysine regioselectivity is selected from the group consisting of: lipases from Candida antarcticis B (i.e., Candida antarcticis lipase B (CALB)), lipases from Aspergillus niger (i.e., Aspergillus niger lipase), lipases from Burkholderia cepacia (i.e., Burkholderia cepacia lipase), and lipases from Rhizopus oryzae (Rhizopus oryzae lipase).

[0050] The enzymatic activity of lipase can be measured using the amidation method fully described in the Examples section below. Lipase activity can be expressed in µmol N-butylacetamide / min / g immobilized lipase. Typically, the lipase used in this invention has an activity of at least 5 µmol N-butylacetamide / min / g immobilized lipase, particularly at least 10 µmol N-butylacetamide / min / g immobilized lipase.

[0051] In the method according to the invention, the protease or lipase is used in an immobilized form, wherein the enzyme is carried on a carrier. There are no limitations on the carrier; it can be any known organic or inorganic material having a suitable shape or configuration.

[0052] In any method according to the invention, the protease or lipase may also be used in a non-immobilized form, wherein the enzyme is not immobilized or is not contained in a solid support. According to any embodiment of the invention, non-immobilized enzymes can be used as alternatives to immobilized enzymes to achieve the same or similar technical effects.

[0053] Suitable supports may include, but are not limited to, inorganic supports selected from the group consisting of: silica, activated carbon, zeolite, alumina, titanium dioxide, zirconium oxide, silica-zirconium-alumina, diatomaceous earth, bentonite, glass, ceramics, and kaolin; and polymer supports selected from the group consisting of: polysaccharides (e.g., agarose, agarose, and cellulose), (meth)acrylic acid polymers (e.g., homopolymers of (meth)acrylates, copolymers of (meth)acrylates, and polyacrylamide), polystyrene optionally crosslinked with divinylbenzene, epoxy resins, polyurethanes, polypropylene, nylon, polyethylene glycol, collagen, starch, and polyvinyl chloride. Supports may also be ion exchange resins.

[0054] For the purposes of this invention, any useful, commercially available immobilized protease and immobilized lipase can be used. Alternatively, the immobilized protease and immobilized lipase can be prepared by suitable methods.

[0055] In some embodiments, immobilized proteases and immobilized lipases can be prepared by a method comprising the following steps: a) preparing a liquid medium containing the protease or lipase in a buffer solution; b) introducing the carrier into the liquid medium, gently stirring for a sufficient period of time, and rebalancing for a sufficient period of time without stirring; and c) separating the resulting supernatant and washing the obtained carrier carrying the protease or lipase. For example, the buffer solution in step a) can be phosphate-buffered saline. For example, the stirring in step b) can be performed for at least 10 hours or at least 15 hours. The equilibration in step b) can be performed for at least 15 hours or at least 18 hours. The carrier can be pretreated by washing prior to step a). The carrier carrying the protease or lipase obtained from step c) can also be washed, for example, with water, sodium chloride solution, or a buffer solution.

[0056] Suitable lysine esters can be, but are not limited to, alkyl or aryl esters of lysine (i.e., L-lysine or D-lysine). According to the invention, L-lysine esters can be used to produce L-polylysine, and D-lysine esters can be used to produce D-polylysine, particularly using L-lysine. Alkyl esters of lysine can, for example, be lysine C... 1-6 -alkyl esters, and lysine aryl esters can be, for example, lysine phenyl esters or benzyl esters.

[0057] Suitable arginine esters may include, but are not limited to, arginine alkyl or aryl esters, such as arginine C 1-6 - Alkyl esters and arginine phenyl esters or benzyl esters.

[0058] In some embodiments, lysine esters are selected from lysine C. 1-6 -Alkyl esters, such as lysine C 1-4-Alkyl esters, such as lysine methyl ester, lysine ethyl ester, lysine propyl ester and lysine butyl ester, of which lysine methyl ester and lysine ethyl ester are particularly useful for the present invention.

[0059] In some other embodiments, arginine esters are selected from arginine C. 1-6 -alkyl esters, such as arginine C 1-4 -Alkyl esters, such as arginine methyl ester, arginine ethyl ester, arginine propyl ester and arginine butyl ester, of which arginine methyl ester and arginine ethyl ester are particularly useful for the present invention.

[0060] Lysine esters and arginine esters can be prepared by any known method or are commercially available. These esters are commercially available in salt form. In this case, the method according to the invention may include desalting the salt of lysine or arginine ester to provide lysine or arginine ester in free base form. The salts of these esters are typically inorganic acid salts, such as hydrochlorides, hydrobromic acids, sulfates, nitrates, or phosphates, with hydrochlorides being particularly suitable for the invention. Suitable salts of these esters may be C 1-6 -Alkyl ester salts, especially C 1-6 -Inorganic acid salts of alkyl esters, especially C 1-6 - Hydrochloride salts of alkyl esters. Suitable salts of these esters may also include organic acid salts, such as formate, acetate, citrate, lactate, fumarate, and propionic acid.

[0061] Desalting can be performed as a separate step prior to polymerization. For example, desalting can be carried out in an aqueous medium with an alkaline pH to separate alkyl esters. A suitable pH may be at least 8.5, or at least 9.0, or at least 10. The alkaline pH can be provided by using a suitable substance (e.g., sodium bicarbonate or sodium hydroxide). In particular, a saturated sodium bicarbonate solution can be used as the aqueous medium with an alkaline pH. Desalting can be carried out at temperatures of 15°C or lower, or 10°C or lower, or 5°C or lower, for example in an ice bath, a jacketed vessel with a circulating coolant, or a cold chamber. The desalting product can be obtained by conventional means, for example by extracting the free base of lysine or arginine ester from the aqueous solution into the organic phase. The treatment of the desalting product can also be carried out at low temperatures of 15°C or lower, or 10°C or lower, or 5°C or lower.

[0062] Desalination can also be performed by ion exchange using a suitable anion exchange resin. Lysine or arginine ester salts can be suspended in a suitable organic solvent (such as chloroform or dichloromethane) and subjected to ion exchange at, for example, 15°C or lower, or 10°C or lower. After ion exchange, the desalination products can be processed using conventional methods.

[0063] Alternatively, in-situ desalting can be employed in the method according to the invention. In-situ desalting can be carried out simply by incorporating an organic base, optionally with a certain amount of water, prior to chemically enzymatic bulk polymerization. Suitable organic bases may include, but are not limited to, any amine that will not adversely affect chemically enzymatic bulk polymerization, such as tertiary amines (e.g., triethylamine, N,N-diisopropylethylamine), tertiary alkyl alcohol amines (e.g., triethanolamine), tertiary diamines (e.g., tetramethylethylenediamine), and aromatic amines (e.g., pyridine).

[0064] As used herein, the term "in-situ desalination" refers to a desalination reaction using an organic base that will remain in the bulk polymerization process without being removed.

[0065] In some specific embodiments, chemically-enzymatic bulk polymerization can be carried out simultaneously with in-situ desalting. In-situ desalting combined with bulk polymerization means that the bulk polymerization is performed in the presence of the organic base used in the desalting reaction without its removal.

[0066] Chemical enzymatic bulk polymerization can be carried out at temperatures ranging from -20°C to 80°C, preferably from 0°C to 35°C. Typically, chemical enzymatic bulk polymerization can be carried out for a period of at least 12 hours, particularly at least 20 hours. Chemical enzymatic bulk polymerization can be carried out for a period of up to 192 hours.

[0067] Polylysine or polyarginine obtained by chemically enzymatic bulk polymerization can be separated by any conventional means. For example, the obtained polylysine or polyarginine can be separated from the immobilized enzyme (i.e., protease or lipase) by washing with a solvent or dissolving it in a solvent and then filtering. The solvent can be water, a suitable organic solvent, or a combination thereof. Further recovery of polylysine or polyarginine obtained from the solvent can be carried out by any conventional means without limitation.

[0068] The inventors have discovered that the immobilized enzyme obtained after separation from polylysine or polyarginine can be used, respectively, for further bulk polymerization of lysine or arginine esters in bulk to regioselectively provide polylysine or polyarginine. In other words, the recycled immobilized enzyme can be further used for the chemically enzymatic polymerization of lysine or arginine esters to regioselectively provide polylysine or polyarginine.

[0069] Therefore, in some embodiments, the method for preparing polylysine or polyarginine according to the present invention may further include:

[0070] - Separate the immobilized enzyme from polylysine or polyarginine, such as those obtained by chemical enzymatic polymerization, and

[0071] - Apply the isolated immobilized enzyme to the additional chemical enzymatic polymerization of lysine or arginine esters.

[0072] It should be understood that the obtained polylysine or polyarginine will have a distribution of polylysine or polyarginine with various degrees of polymerization within a certain range. The method according to the invention can provide polylysine with a degree of polymerization (DP) ranging from 2 to 60, particularly from 2 to 50. For example, the degree of polymerization (DP) range of polylysine obtained by the method according to the invention can have a peak maximum DP in the range of 4 to 25, particularly from 6 to 20.

[0073] In embodiments where an immobilized protease is used to regioselectively provide α-polylysine, the obtained polylysine has a DP range of 2 to 60, particularly 2 to 50, with a peak maximum of 4 to 15. The regioselectivity of α-polylysine can be at least 60%, preferably at least 70%, and more preferably at least 80%.

[0074] In embodiments where immobilized lipase is used to regioselectively provide ε-polylysine, the obtained polylysine has a DP range of 2 to 50, particularly 2 to 40, with a peak maximum of 4 to 10. The regioselectivity of ε-polylysine can be at least 70%, preferably at least 80%, and more preferably at least 85%.

[0075] The method according to the invention can provide polyarginine with a degree of polymerization (DP) ranging from 2 to 30, particularly from 2 to 20. The obtained polyarginine may have a peak maximum DP in the range of 2 to 5. The regioselectivity of α-polyarginine may be at least 75%, preferably at least 90%, more preferably at least 95%.

[0076] The inventors have unexpectedly discovered that polylysine with high α-regioselectivity or ε-regioselectivity and polyarginine with high α-regioselectivity can be successfully synthesized by chemical enzymatic bulk polymerization in the presence of the corresponding enzymes.

[0077] The inventors have unexpectedly discovered that polylysine with high α-regioselectivity or ε-regioselectivity and polyarginine with high α-regioselectivity can be successfully synthesized by chemical enzymatic bulk polymerization in the presence of corresponding immobilized or non-immobilized enzymes.

[0078] Therefore, in a second aspect, the present invention further provides a method for controlling the regioselectivity of linear polylysine or polyarginine in the chemical enzymatic polymerization of lysine esters or arginine esters in bulk, wherein an immobilized protease is used to regioselectively provide α-polylysine or α-polyarginine, or an immobilized lipase is used to regioselectively provide ε-polylysine.

[0079] In the method of this invention, the lysine ester can be, but is not limited to, an alkyl or aryl ester of lysine (i.e., L-lysine or D-lysine). The alkyl or aryl ester of lysine can be, for example, a lysine C-ester. 1-6 -Alkyl esters, and lysine aryl esters can be, for example, lysine phenyl esters or benzyl esters. Arginine esters can include, but are not limited to, arginine alkyl or aryl esters, such as arginine C 1-6 - Alkyl esters and arginine phenyl esters or benzyl esters.

[0080] In some embodiments, lysine esters are selected from lysine C. 1-6 -Alkyl esters, such as lysine C 1-4 -Alkyl esters, such as lysine methyl ester, lysine ethyl ester, lysine propyl ester and lysine butyl ester, wherein lysine methyl ester and lysine ethyl ester are particularly useful for the present invention.

[0081] In some other embodiments, arginine esters are selected from arginine C. 1-6 -alkyl esters, such as arginine C 1-4 -Alkyl esters, such as arginine methyl ester, arginine ethyl ester, arginine propyl ester and arginine butyl ester, of which arginine methyl ester and arginine ethyl ester are particularly useful for the present invention.

[0082] Suitable proteases for catalyzing peptide formation reactions selectively toward the α-polylysine or α-polyarginine region may include, but are not limited to, serine proteases (e.g., trypsin, α-chymotrypsin, elastase, carboxypeptidase, and subtilisin), cysteine ​​proteases (e.g., papain, fig protease, bromelain, annalin, calpain, caspase, papain, cathepsin, clostridium protease, and actinidin), metalloproteinases (e.g., thermophilic proteases, collagenases, bacillus lysins, dispersins, vibrio lysins, pseudolysins, matrix lysins, and various bacterial-derived neutral metalloproteinases), aspartic proteases (e.g., pepsin, penicillin pepsin, chymotrypsin, cathepsin, renin, β-secretase, Plasmodium plasma proteases, and retroviral proteases), liver esterases (e.g., porcine liver esterase), alkaline proteases, carbonic anhydrases, nonribosomal peptide synthases, thrombin, carboxylic acid A or B, or streptomycin).

[0083] In some embodiments, the protease used to catalyze the peptide formation reaction selectively toward the α-polylysine or α-polyarginine region is selected from the group consisting of serine proteases (such as trypsin, α-chymotrypsin, elastase, carboxypeptidase, and subtilisin) and cysteine ​​proteases (such as papain, figolytic enzyme, bromelain, annalin, calpain, caspase, papain, cathepsin, clostridium protease, and actinidin). Specifically, the protease is selected from the group consisting of trypsin, subtilisin, and papain.

[0084] Suitable lipases for catalyzing peptide formation reactions with selectivity toward the ε-polylysine region may include, but are not limited to, lipases (EC3.1.1.3), such as lipases from Candida antarctica B, and lipases from species of Alcaligenes, Aspergillus, Mucor, Penicillium, Geotrichum, Rhizopus, Burkholderia, Candida, Pseudomonas, and Thermophilus.

[0085] In some embodiments, the lipase used to catalyze the peptide formation reaction selectively toward the ε-polylysine region is selected from the group consisting of: lipases from Candida antarctica B (i.e., Candida antarctica lipase B (CALB)), lipases from Aspergillus niger (i.e., Aspergillus niger lipase), lipases from Burkholderia cepacia (i.e., Burkholderia cepacia lipase), and lipases from Rhizopus oryzae (Rhizopus oryzae lipase).

[0086] Suitable carriers for immobilizing proteases and immobilized lipases are those described above in the context of the method for preparing polylysine or polyarginine in the first aspect of the invention.

[0087] In some embodiments according to the second aspect, the present invention relates to a method for separately processing lysine C 1-6 -Alkyl esters or arginine C 1-6 A method for controlling the regioselectivity of linear polylysine or polyarginine in the chemical enzymatic polymerization of α-alkyl esters in bulk, wherein an immobilized protease selected from the group consisting of serine proteases (such as trypsin and subtilisin) and cysteine ​​proteases (such as papain) is used to regioselectively provide α-polylysine or α-polyarginine.

[0088] In some embodiments according to the second aspect, the present invention relates to a method for separately processing lysine C 1-6 -Alkyl esters or arginine C 1-6A method for controlling the regioselectivity of linear polylysine or polyarginine in the chemical enzymatic polymerization of α-alkyl esters in bulk, wherein immobilized or non-immobilized proteases selected from the group consisting of serine proteases (such as trypsin and subtilisin) and cysteine ​​proteases (such as papain) are used to regioselectively provide α-polylysine or α-polyarginine.

[0089] In some other embodiments according to the second aspect, the present invention relates to a method for [using] lysine C 1-6 A method for controlling the regioselectivity of linear polylysine in the chemi-enzymatic polymerization of alkyl esters in bulk, wherein an immobilized lipase selected from the group consisting of Candida antarcticis lipase B, Aspergillus niger lipase, Burkholderia cepacia lipase and Rhizopus oryzae lipase is used to regioselectively provide ε-polylysine.

[0090] In some other embodiments according to the second aspect, the present invention relates to a method for [using] lysine C 1-6 A method for controlling the regioselectivity of linear polylysine in the chemi-enzymatic polymerization of alkyl esters in bulk, wherein immobilized or non-immobilized lipases selected from the group consisting of Candida antarcticis lipase B, Aspergillus niger lipase, Burkholderia cepacia lipase and Rhizopus oryzae lipase are used to regioselectively provide ε-polylysine.

[0091] Chemical enzymatic bulk polymerization can be carried out at temperatures ranging from -20°C to 80°C, preferably from 0°C to 35°C. Typically, chemical enzymatic bulk polymerization can be carried out for a period of at least 12 hours, particularly at least 20 hours. Chemical enzymatic bulk polymerization can be carried out for a period of up to 192 hours.

[0092] According to some embodiments of the present invention, chemically enzymatic bulk polymerization can be carried out by adding an additional polar organic solvent, wherein the polar organic solvent is selected from the group consisting of: acetonitrile, dimethyl sulfoxide, acetone, methyl acetate, ethyl acetate, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, propylene carbonate, dichloromethane, chloroform, sulfolane, hexamethylphosphoramide, 1,3-dimethyl-2-imidazolinone, nitromethane, butanone, methanol, methylformamide, ethanol, isopropanol, 1-propanol, tert-butanol, formamide, n-butanol, ethylene glycol, 2-methoxyethanol, 1-methoxy-2-propanol, glycerol, 1,2-propanediol and 1,3-propanetriol, and toluene.

[0093] The enzymatic activity of proteases, particularly trypsin, can be measured using the Nα-benzoyl-DL-arginine-p-nitroaniline (BAPNA) assay, as fully described in the Examples section below. The trypsin used in this invention has an activity of at least 0.05 U / g, preferably at least 0.1 U / g, and more preferably at least 0.3 U / g.

[0094] In some embodiments of the invention, the enzyme activity of the non-immobilized protease (natural protease) is, for example, 10 U / mg to 50 U / mg, preferably 20 U / mg to 40 U / g. In some embodiments of the invention, the enzyme activity of the non-immobilized protease is, for example, 500 USP / mg to 5000 USP / mg, for example 800 USP / mg to 4500 USP / mg.

[0095] The enzymatic activity of lipase can be measured using the amidation method fully described in the Examples section below. Lipase activity can be expressed in µmol N-butylacetamide / min / g immobilized lipase. Typically, the lipase used in this invention has an activity of at least 5 µmol N-butylacetamide / min / g immobilized lipase, particularly at least 10 µmol N-butylacetamide / min / g immobilized lipase.

[0096] In the method of the present invention, the regioselectivity of α-polylysine can be at least 60%, preferably at least 70%, more preferably at least 80%. The regioselectivity of ε-polylysine can be at least 70%, preferably at least 80%, more preferably at least 85%. The regioselectivity of α-polyarginine can be at least 75%, preferably at least 90%, more preferably at least 95%. Example

[0097] Various embodiments are listed below. It will be understood that the embodiments listed below can be combined with all aspects of the invention and other embodiments.

[0098] Example 1: A method for preparing polylysine or polyarginine, the method comprising chemically enzymatically polymerizing lysine esters in bulk in the presence of immobilized or non-immobilized proteases to regioselectively provide α-polylysine, or chemically enzymatically polymerizing lysine esters in the presence of immobilized or non-immobilized lipases to regioselectively provide ε-polylysine, or chemically enzymatically polymerizing arginine esters in bulk in the presence of immobilized or non-immobilized proteases to regioselectively provide α-polyarginine.

[0099] Example 2: The method according to Example 1, wherein the lysine ester is selected from the group consisting of lysine alkyl esters and lysine aryl esters, and the arginine ester is selected from the group consisting of arginine alkyl esters and arginine aryl esters.

[0100] Example 3: The method according to Example 2, wherein the lysine ester is selected from lysine C. 1-6 -Alkyl ester.

[0101] Example 4: The method according to Example 3, wherein the lysine ester is selected from lysine C. 1-4 -Alkyl ester.

[0102] Example 5: The method according to Example 4, wherein the lysine ester is selected from lysine methyl ester, lysine ethyl ester, lysine propyl ester and lysine butyl ester.

[0103] Example 6: The method according to Example 2, wherein the lysine ester is selected from lysine phenyl ester and lysine benzyl ester.

[0104] Example 7: The method according to Example 2, wherein the arginine ester is selected from arginine C. 1-6 -Alkyl ester.

[0105] Example 8: The method according to Example 7, wherein the arginine ester is selected from arginine C. 1-4 -Alkyl ester.

[0106] Example 9: The method according to Example 8, wherein the arginine ester is selected from arginine methyl ester, arginine ethyl ester, arginine propyl ester and arginine butyl ester.

[0107] Example 10: The method according to Example 2, wherein the arginine ester is selected from arginine phenyl ester and arginine benzyl ester.

[0108] Example 11: The method according to any one of the foregoing examples, wherein the protease is selected from the group consisting of serine proteases and cysteine ​​proteases.

[0109] Example 12: The method according to Example 11, wherein the protease is selected from the group consisting of: trypsin, α-chymotrypsin, elastase, carboxypeptidase and subtilisin.

[0110] Example 13: The method according to Example 11, wherein the protease is selected from the group consisting of: papain, fig protease, bromelain, annalin, calpain, caspase, papain chymopapain, cathepsin, clostridium protease and actinidin.

[0111] Example 14: The method according to Example 11, wherein the protease is selected from the group consisting of trypsin, subtilisin and papain.

[0112] Example 15: The method according to any one of Examples 1 to 10 above, wherein the lipase is selected from lipases (EC 3.1.1.3).

[0113] Example 16: The method according to Example 15, wherein the lipase is selected from the group consisting of: lipase from Candida antarcticis B (i.e., Candida antarcticis lipase B (CALB)), lipase from Aspergillus niger (i.e., Aspergillus niger lipase), lipase from Burkholderia cepacia (i.e., Burkholderia cepacia lipase) and lipase from Rhizopus oryzae (Rhizopus oryzae lipase).

[0114] Example 17: The method according to any one of the foregoing examples further includes desalting the salt of the lysine ester to provide the lysine ester, or desalting the salt of the arginine ester to provide the arginine ester.

[0115] Example 18: The method according to Example 17, wherein the salt of the lysine ester is an inorganic acid salt of the lysine ester.

[0116] Example 19: The method according to Example 17, wherein the salt of the arginine ester is an inorganic acid salt of the arginine ester.

[0117] Example 20: The method according to any one of the preceding examples, wherein the chemical enzymatic polymerization is carried out at a temperature in the range of -20°C (negative 20°C) to 80°C.

[0118] Example 21: The method according to Example 20, wherein the chemical enzymatic polymerization is carried out at a temperature in the range of 0°C to 35°C.

[0119] Example 22: The method according to any one of the preceding examples, wherein the chemical enzymatic polymerization is carried out for a period of at least 12 hours, particularly at least 20 hours.

[0120] Example 23: The method according to any one of the foregoing examples, wherein the chemical enzymatic polymerization is carried out for a period of up to 192 hours.

[0121] Example 24: The method according to any one of the foregoing examples further includes separating the immobilized enzyme or the non-immobilized enzyme from the polylysine or the polyarginine obtained from the chemical enzymatic polymerization.

[0122] Example 25: The method according to Example 24 further includes applying the immobilized enzyme or the non-immobilized enzyme from the isolated enzyme to additional chemical enzymatic polymerization of lysine esters or arginine esters.

[0123] Example 26: The method according to any one of the preceding examples, wherein the polylysine has a degree of polymerization in the range of 2 to 60, and the polyarginine has a degree of polymerization in the range of 2 to 30.

[0124] Example 27: The method according to Example 26, wherein the polylysine has a degree of polymerization in the range of 2 to 50, and the polyarginine has a degree of polymerization in the range of 2 to 20.

[0125] Example 28: The method according to Example 26 or 27, wherein the degree of polymerization of the polylysine has a peak maximum value in the range of 4 to 25, and the degree of polymerization of the polyarginine has a peak maximum value in the range of 2 to 5.

[0126] Example 29: The method according to Example 28, wherein the degree of polymerization of the polylysine has a peak maximum value in the range of 6 to 20.

[0127] Example 30: A method for controlling the regioselectivity of linear polylysine or polyarginine in the chemical enzymatic polymerization of lysine esters or arginine esters in bulk, wherein an immobilized or non-immobilized protease is used to regioselectively provide α-polylysine or α-polyarginine, or an immobilized or non-immobilized lipase is used to regioselectively provide ε-polylysine.

[0128] Example 31: The method according to Example 30, wherein the lysine ester is selected from the group consisting of lysine alkyl esters and lysine aryl esters, and the arginine ester is selected from the group consisting of arginine alkyl esters and arginine aryl esters.

[0129] Example 32: The method according to Example 31, wherein the lysine ester is selected from lysine C. 1-6 -Alkyl ester.

[0130] Example 33: The method according to Example 32, wherein the lysine ester is selected from lysine C. 1-4 -Alkyl ester.

[0131] Example 34: The method according to Example 33, wherein the lysine ester is selected from lysine methyl ester, lysine ethyl ester, lysine propyl ester and lysine butyl ester.

[0132] Example 35: The method according to Example 31, wherein the lysine ester is selected from lysine phenyl and lysine benzyl ester.

[0133] Example 36: The method according to Example 31, wherein the arginine ester is selected from arginine C. 1-6 -Alkyl ester.

[0134] Example 37: The method according to Example 36, wherein the arginine ester is selected from arginine C. 1-4 -Alkyl ester.

[0135] Example 38: The method according to Example 37, wherein the arginine ester is selected from arginine methyl ester, arginine ethyl ester, arginine propyl ester and arginine butyl ester.

[0136] Example 39: The method according to Example 31, wherein the arginine ester is selected from arginine phenyl ester and arginine benzyl ester.

[0137] Example 40: The method according to any one of Examples 30 to 39, wherein the protease is selected from the group consisting of serine proteases and cysteine ​​proteases.

[0138] Example 41: The method according to Example 40, wherein the protease is selected from the group consisting of: trypsin, α-chymotrypsin, elastase, carboxypeptidase and subtilisin.

[0139] Example 42: The method according to Example 40, wherein the protease is selected from the group consisting of: papain, fig protease, bromelain, annalin, calpain, caspase, papain chymopapain, cathepsin, clostridium protease and actinidin.

[0140] Example 43: The method according to Example 40, wherein the protease is selected from the group consisting of trypsin, subtilisin and papain.

[0141] Example 44: The method according to any one of Examples 30 to 39, wherein the lipase is selected from lipases (EC 3.1.1.3).

[0142] Example 45: The method according to Example 44, wherein the lipase is selected from the group consisting of: lipase from Candida antarcticis B (i.e., Candida antarcticis lipase B (CALB)), lipase from Aspergillus niger (i.e., Aspergillus niger lipase), lipase from Burkholderia cepacia (i.e., Burkholderia cepacia lipase) and lipase from Rhizopus oryzae (Rhizopus oryzae lipase).

[0143] Example 46: The method according to any one of Examples 30 to 45, wherein the chemical enzymatic polymerization is carried out at a temperature in the range of -20°C (negative 20°C) to 80°C, preferably in the range of 0°C to 35°C.

[0144] Example 47: The method according to any one of Examples 30 to 46, wherein the chemical enzymatic polymerization is carried out for a period of at least 12 hours, particularly at least 20 hours, and preferably up to 192 hours.

[0145] Example 48: The method according to any one of Examples 30 to 47, wherein the regioselectivity of α-polylysine is at least 60%, preferably at least 70%.

[0146] Example 49: The method according to any one of Examples 30 to 47, wherein the regioselectivity of ε-polylysine may be at least 70%, preferably at least 80%.

[0147] Example 50: The method according to any one of Examples 30 to 47, wherein the regioselectivity of ε-polyarginine may be at least 75%, preferably at least 90%. Example

[0148] The various aspects of the invention will be more fully illustrated by the following examples, which are presented to illustrate certain aspects of the invention and should not be construed as limiting it.

[0149] Enzyme activity measurement

[0150] The activity of the immobilized enzymes used in the following examples was measured according to the corresponding procedures described below.

[0151] Measurement of immobilized lipase activity

[0152] The activity of the immobilized lipase used for the chemical enzymatic bulk polymerization of lysine esters was estimated by using the activity of the immobilized lipase in the reaction of n-butylamine with benzyl acetate to form n-butylacetamide, as shown below.

[0153]

[0154] 50 µL of benzyl acetate and 50 µL of n-butylamine were reacted at 25°C in the presence of 20 mg of immobilized lipase for 60 min, and then... 1 Conversion was determined by ¹H NMR (without additional solvent). The amount of N-butylacetamide produced per minute per gram of immobilized lipase (expressed as µmol N-butylacetamide / min / g) was reported as the activity of the immobilized lipase.

[0155]

[0156] Activity measurement of immobilized trypsin

[0157] The activity of immobilized trypsin was estimated using an Nα-benzoyl-DL-arginine-p-nitroaniline (BAPNA) assay. Enzyme activity was measured using absorbance at 410 nm. One unit (U) of enzyme activity was defined as the activity at pH 8.2 and 25°C in Ca2+. 2+ The amount of enzyme capable of hydrolyzing 1 µmol of BAPNA per minute in the presence of [specific enzyme name]. The amount of BAPNA hydrolyzed per minute by each gram of immobilized trypsin (expressed as U / g) is reported as the activity of the immobilized trypsin.

[0158]

[0159] The activity of commercially available immobilized trypsin purchased from Thermo Fisher Scientific is 0.812 U / g. The activity of the prepared immobilized trypsin is provided in the corresponding examples.

[0160] Example 1

[0161] Lysine ethyl ester (Lys-OEt) was prepared by desalting Lys-OEt dihydrochloride.

[0162] 5 g of lysine ethyl ester dihydrochloride (Lys-OEt dihydrochloride) was dissolved in a saturated sodium bicarbonate solution in an ice bath. The pH was then adjusted to 10.5 by adding 5 M sodium hydroxide (NaOH) solution. The resulting aqueous solution was poured into a 300 mL extraction funnel to extract Lys-OEt from the solution by shaking with excess chloroform, followed by phase separation. All materials were pre-cooled to inhibit thermal condensation of Lys-OEt during extraction, and the entire process was carried out in a cold chamber. Finally, chloroform was evaporated at 10°C using a vacuum pump to produce liquid Lys-OEt.

[0163] Ontological Aggregation of Lys-OEt

[0164] 200 µL of Lys-OEt was poured into a polypropylene microtube, and 10 mg of acrylic resin with immobilized CALB (purchased from Sigma-Aldrich) was added. Bulk polymerization was carried out at 40°C for 24 h. The mixture was then cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-coupled time-of-flight mass spectrometry (MALDI-TOF MS) and... 1 Characterization was performed using ¹H NMR to determine the degree of polymerization (DP) range, monomer conversion, and regioselectivity. The DP range was 2 to 11, with a peak maximum of 4, a conversion of 37%, and a regioselectivity (ε) of 72%.

[0165] Example 2

[0166] The method was performed according to Example 1, except that 20 mg of acrylic resin with immobilized CALB (purchased from Sigma-Aldrich) was used for bulk polymerization. The DP range was 2 to 12, with a peak maximum of 5, the conversion was 42%, and the regioselectivity (ε) was 77%.

[0167] Example 3

[0168] The method was performed according to Example 1, except that 30 mg of acrylic resin with immobilized CALB (purchased from Sigma-Aldrich) was used for polymerization. The DP range was 2 to 13, with a peak maximum of 5, the conversion was 47%, and the regioselectivity (ε) was 85%.

[0169] Example 4

[0170] The method was performed according to Example 3, except that the temperature was maintained at 30°C during bulk polymerization. The DP range was 2 to 12, with a peak maximum of 4, the conversion rate was 32%, and the regioselectivity (ε) was 88%.

[0171] Example 5

[0172] The method was performed according to Example 3, except that the temperature was maintained at 20°C during bulk polymerization. The DP range was 2 to 16, with a peak maximum of 6, the conversion rate was 61%, and the regioselectivity (ε) was 92%.

[0173] Example 6

[0174] The method was performed according to Example 3, except that the temperature was maintained at 10°C during bulk polymerization. The DP range was 2 to 12, with a peak maximum of 4, the conversion rate was 56%, and the regioselectivity (ε) was 96%.

[0175] Example 7

[0176] 200 µL of Lys-OEt, prepared as in Example 1, was poured into a polypropylene microtube, and 10 mg of Amano lipase PS (lipase immobilized on diatomaceous earth, purchased from Sigma-Aldrich) was added. Bulk polymerization was carried out at 10°C for 72 h. The mixture was then cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 2 to 12, with a peak maximum of 4, conversion was 54%, and regioselectivity (ε) was 73%.

[0177] Example 8

[0178] Lysine ethyl ester (Lys-OEt) was prepared by desalting Lys-OEt dihydrochloride.

[0179] 5 g of Lys-OEt dihydrochloride was suspended in 50 ml of chloroform under vigorous stirring. Then, 20 g of strong anion exchange resin Amberlite IRN78 (purchased from Sigma-Aldrich) was added, and the mixture was equilibrated at 10°C with gentle stirring for 30 min. All materials were pre-cooled to inhibit thermal condensation of Lys-OEt during extraction, and the entire process was carried out in a cold chamber. Finally, the exchange resin was filtered off, and the chloroform was evaporated using a vacuum pump at 10°C to produce liquid Lys-OEt.

[0180] Ontological Aggregation of Lys-OEt

[0181] 200 µL of Lys-OEt was poured into a polypropylene microtube, and 30 mg of acrylic resin with immobilized CALB (purchased from Sigma-Aldrich) was added. Bulk polymerization was carried out at 10°C for 72 h. The mixture was then cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 3 to 22, with a peak maximum of 6, conversion was 64%, and regioselectivity (ε) was 99%.

[0182] Example 9

[0183] The method was performed according to Example 6, except that the ontological aggregation was carried out for 96 hours. The DP range was 2 to 15, with a peak maximum of 5, the conversion rate was 68%, and the regioselectivity (ε) was 99%.

[0184] Example 10

[0185] Lysine ethyl ester (Lys-OEt) was prepared by desalting Lys-OEt dihydrochloride.

[0186] 30 g of Lys-OEt dihydrochloride was suspended in 300 ml of chloroform under vigorous stirring. Then, 120 g of Amberlite IRN78 strong anion exchange resin (purchased from Sigma-Aldrich) was added, and the mixture was equilibrated at 4°C with gentle stirring for 60 min. All materials were pre-cooled to inhibit thermal condensation of Lys-OEt during extraction, and the entire process was carried out in a cold chamber. The resin was filtered off, and the solution was dried over anhydrous NaSO4. Finally, chloroform was evaporated at 10°C using a vacuum pump to produce liquid Lys-OEt.

[0187] Ontological Aggregation of Lys-OEt

[0188] Lys-OEt was polymerized in the ontology according to Example 8. The DP range was 2 to 19, with a peak maximum of 6, the conversion rate was 100%, and the regioselectivity (ε) was 96%.

[0189] Example 11

[0190] 200 µL of Lys-OEt, prepared as in Example 1, was poured into a polypropylene microtube, and 30 mg of Novozym435 (CALB immobilized on the polymer resin, purchased from Novozymes) was added. Bulk polymerization was carried out at 10°C for 72 h. The mixture was then cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 2 to 16, with a peak maximum of 5, conversion was 100%, and regioselectivity (ε) was 100%.

[0191] Example 12

[0192] The method was performed according to Example 11, except that 60 mg of Novozym 435 (CALB immobilized on the polymer resin, purchased from Novozymes) was used for polymerization. The DP range was 3 to 23, with a peak maximum of 6, the conversion was 100%, and the regioselectivity (ε) was 100%.

[0193] Example 13

[0194] 200 µL of Lys-OEt, prepared as in Example 1, was poured into a polypropylene microtube, and 30 mg of Amano lipase CLI IM (lipase immobilized on polyacrylate resin, available from Amano Enzyme Co., Ltd.) was added. Bulk polymerization was carried out at 10°C for 72 h. The mixture was then cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 3 to 28, with a peak maximum of 5, conversion was 100%, and regioselectivity (ε) was 100%.

[0195] Example 14

[0196] 200 µL of Lys-OEt, prepared as in Example 1, was poured into a polypropylene microtube. Then, 10 mg of agarose beads (purchased from Thermo Fisher Scientific) pre-washed five times with water and immobilized trypsin were added to the microtube. Bulk polymerization was carried out at 40°C for 24 h. The mixture was then cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1Characterized by 1H NMR. DP ranged from 2 to 8, with a peak maximum of 4, conversion was 79%, and regioselectivity (α) was 64%.

[0197] Example 15

[0198] The method was performed according to Example 14, except that 30 mg of agarose beads with immobilized trypsin (purchased from Thermo Fisher Scientific) were used for polymerization. The DP range was 2 to 8, with a peak maximum of 4, the conversion was 63%, and the regioselectivity (α) was 75%.

[0199] Example 16

[0200] 200 µL of Lys-OEt, prepared as in Example 1, was poured into a polypropylene microtube. Then, 30 mg of agarose beads (purchased from Thermo Fisher Scientific) pre-washed five times with water and immobilized trypsin were added to the microtube. Bulk polymerization was carried out at 20°C for 72 h. The mixture was then cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 3 to 22, with a peak maximum of 8, conversion was 69%, and regioselectivity (α) was 64%.

[0201] Example 17

[0202] 200 µL of Lys-OEt, prepared according to Example 1, was poured into a polypropylene microtube. Subsequently, 30 mg of agarose beads (purchased from Thermo Fisher Scientific) pre-washed five times with water and immobilized trypsin were added to the microtube. Bulk polymerization was carried out at 10°C for 168 h, with crude products collected at 48 h, 72 h, and 168 h. The final mixture was cooled to -30°C to stop the reaction. The crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. The results are summarized in Table 1.

[0203] Table 1

[0204]

[0205] Example 18

[0206] 200 µL of Lys-OEt, prepared as in Example 1, was poured into a polypropylene microtube. Subsequently, 60 mg of agarose beads (purchased from Thermo Fisher Scientific) pre-washed five times with water and immobilized trypsin were added to the microtube. Bulk polymerization was carried out at 10°C for 72 h. The mixture was then cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 4 to 27, with a peak maximum of 10, conversion was 57%, and regioselectivity (α) was 87%.

[0207] Example 19

[0208] The method was performed according to Example 18, except that 90 mg of agarose beads with immobilized trypsin (purchased from Thermo Fisher Scientific) were used for polymerization. The DP range was 5 to 30, with a peak maximum of 11, the conversion rate was 45%, and the regioselectivity (α) was 90%.

[0209] Example 20

[0210] 200 µL of Lys-OEt, prepared as in Example 1, was poured into a polypropylene microtube. Then, 120 mg of agarose beads (purchased from Thermo Fisher Scientific) pre-washed five times with water and immobilized trypsin were added to the microtube. Bulk polymerization was carried out at 30°C for 72 h. The mixture was then cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 2 to 26, with a peak maximum of 5, conversion was 89%, and regioselectivity (α) was 82%.

[0211] Example 21

[0212] The method was performed according to Example 20, except that the temperature was maintained at 20°C during bulk polymerization. The DP range was 2 to 27, with a peak maximum of 8, the conversion rate was 90%, and the regioselectivity (α) was 86%.

[0213] Example 22

[0214] 200 µL of Lys-OEt, prepared according to Example 1, was poured into a polypropylene microtube. Subsequently, 120 mg of agarose beads (purchased from Thermo Fisher Scientific) pre-washed five times with water and immobilized trypsin were added to the microtube. Bulk polymerization was carried out at 10°C for 96 h, with crude products collected at 24 h, 48 h, and 72 h. The final mixture was cooled to -30°C to stop the reaction. The crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. The results are summarized in Table 2.

[0215] Table 2

[0216]

[0217] Example 23

[0218] The method was performed according to Example 20, except that the temperature was maintained at 4°C during bulk polymerization. The DP range was 2 to 33, with a peak maximum of 9, the conversion rate was 87%, and the regioselectivity (α) was 95%.

[0219] Example 24

[0220] Recycled immobilized trypsin

[0221] The recyclability of the immobilized trypsin was tested by bulk polymerization at 10°C for 72 h according to Example 22. After the initial polymerization, the product was washed with 1000 µL of water, centrifuged at 6000 rpm for 1 min, and the supernatant containing the resulting polylysine was removed. This washing step was repeated five times. Then, 200 µL of Lys-OEt was added to start the next polymerization cycle. Six repeated polymerization cycles were performed. The polymers from each cycle were characterized. The results are summarized in Table 3.

[0222] Table 3

[0223]

[0224] Example 25

[0225] Lysine ethyl ester (Lys-OEt) was prepared by desalting Lys-OEt dihydrochloride.

[0226] 30 g of Lys-OEt dihydrochloride was suspended in 300 ml of chloroform under vigorous stirring. Then, 120 g of strong anion exchange resin Amberlite IRN78 (purchased from Sigma-Aldrich) was added, and the mixture was equilibrated at 4°C with gentle stirring for 60 min. All materials were pre-cooled to inhibit thermal condensation of Lys-OEt during extraction, and the entire process was carried out in a cold chamber. Finally, the exchange resin was filtered off, and the chloroform was evaporated using a vacuum pump at 10°C to produce liquid Lys-OEt.

[0227] Ontological Aggregation of Lys-OEt

[0228] Lys-OEt was polymerized in the ontology according to Example 22, except that the polymerization was carried out for 72 h. The DP range was 3 to 31, with a peak maximum of 10, the conversion rate was 81%, and the regioselectivity (α) was 89%.

[0229] Example 26

[0230] Immobilization of trypsin

[0231] 500 mg of polymeric resin ECR8209F (Purolite) was washed with water at a resin:water ratio of 1:2 w / v, and excess liquid was filtered off. The washing was repeated four times. The washed resin was added to a solution of 25 mg / ml trypsin (1:250, from porcine pancreas, for cell culture, Wako) in 1X phosphate-buffered saline (pH 7.4), where the resin:buffer ratio was 1:4 w / v. The resulting slurry was gently mixed for 18 h using a tubular mixer and then reequilibrated for 20 h without mixing. The supernatant was then collected, and the resin was washed four times with water under gentle mixing. The enzyme immobilization yield was 71%, estimated based on the residual trypsin concentration in the supernatant using a dicaprynic acid assay (BCA assay). The activity of the immobilized trypsin was 1.16 U / g, which was estimated using Nα-benzoyl-DL-arginine-p-nitroaniline (BAPNA).

[0232] Ontological Aggregation of Lys-OEt

[0233] 200 µL of Lys-OEt, prepared as per Example 25, was poured into a polypropylene microtube. Subsequently, 84 mg of resin ECR8209F containing trypsin immobilized according to the above procedure was added to the microtube. Bulk polymerization was carried out at 10°C for 72 h. Afterward, the mixture was cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1Characterized by 1H NMR. DP ranged from 3 to 46, with a peak maximum of 10, conversion was 87%, and regioselectivity (α) was 85%.

[0234] Example 27

[0235] Immobilization of trypsin

[0236] Trypsin was immobilized on polymer resin ECR8209F (Purilet) according to the procedure in Example 26, except that the concentration of trypsin in the immobilization buffer was 12.5 mg / ml. The enzyme immobilization yield was 69%, and the enzyme activity was 0.696 U / g.

[0237] Ontological Aggregation of Lys-OEt

[0238] 200 µL of Lys-OEt, prepared as in Example 25, was poured into a polypropylene microtube. Subsequently, 140 mg of resin ECR8209F containing trypsin immobilized according to the above procedure was added to the microtube. Bulk polymerization was carried out at 10°C for 72 h. Afterward, the mixture was cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 3 to 46, with a peak maximum of 11, conversion was 96%, and regioselectivity (α) was 92%.

[0239] Example 28

[0240] Immobilization of trypsin

[0241] Trypsin was immobilized on polymer resin ECR8285 (Purilet) according to the procedure in Example 27, except that ECR8285 polymer resin was used. The enzyme immobilization yield was 86%, and the enzyme activity was 0.000968 U / g.

[0242] Ontological Aggregation of Lys-OEt

[0243] 200 µL of Lys-OEt, prepared as per Example 25, was poured into a polypropylene microtube. Subsequently, 420 mg of resin ECR8285 containing trypsin immobilized according to the above procedure was added to the microtube. Bulk polymerization was carried out at 10°C for 72 h. Afterward, the mixture was cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 3 to 42, with a peak maximum of 9, conversion was 83%, and regioselectivity (α) was 84%.

[0244] Example 29

[0245] Immobilization of trypsin

[0246] Trypsin was immobilized on Chromalite MEP / M polymer resin (Purilet Corporation) according to the procedure in Example 27, except that Chromalite MEP / M resin was used. The enzyme immobilization yield was 40%, and the enzyme activity was 0.348 U / g.

[0247] Ontological Aggregation of Lys-OEt

[0248] 200 µL of Lys-OEt, prepared as per Example 25, was poured into a polypropylene microtube. Subsequently, 280 mg of Chromalite MEP / M resin containing trypsin immobilized according to the above procedure was added to the microtube. Bulk polymerization was carried out at 10°C for 72 h. The mixture was then cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 3 to 41, with a peak maximum of 10, conversion was 85%, and regioselectivity (α) was 88%.

[0249] Example 30

[0250] Immobilization of trypsin

[0251] Trypsin was immobilized on polymer resin ECR8209F (Purilet) according to the procedure in Example 26, except that 2X phosphate-buffered saline (pH 7.4) was used to dissolve the trypsin. The enzyme immobilization yield was 67%, and the enzyme activity was 0.784 U / g.

[0252] Ontological Aggregation of Lys-OEt

[0253] 200 µL of Lys-OEt, prepared as per Example 25, was poured into a polypropylene microtube. Subsequently, 79 mg of resin ECR8209F containing trypsin immobilized according to the above procedure was added to the microtube. Bulk polymerization was carried out at 10°C for 72 h. Afterward, the mixture was cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 3 to 39, with a peak maximum of 10, conversion was 90%, and regioselectivity (α) was 92%.

[0254] Example 31

[0255] Immobilization of trypsin

[0256] Trypsin was immobilized on polymer resin ECR8209F (Purilet) according to the procedure in Example 26, except that the concentration of trypsin in the immobilization buffer was 62.5 mg / ml. The enzyme immobilization yield was 53%, and the enzyme activity was 0.232 U / g.

[0257] Ontological Aggregation of Lys-OEt

[0258] 200 µL of Lys-OEt, prepared as per Example 25, was poured into a polypropylene microtube. Subsequently, 255 mg of resin ECR8209F containing trypsin immobilized according to the above procedure was added to the microtube. Bulk polymerization was carried out at 10°C for 72 h. Afterward, the mixture was cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 3 to 33, with a peak maximum of 7, conversion was 97%, and regioselectivity (α) was 88%.

[0259] Example 32

[0260] Immobilization of trypsin

[0261] Trypsin was immobilized on polymer resin ECR8209F (Purilet) according to the procedure in Example 26, except that 10 g of ECR8209F resin was used. The enzyme immobilization yield was 49%, and the enzyme activity was 0.420 U / g.

[0262] Ontological Aggregation of Lys-OEt

[0263] 200 µL of Lys-OEt, prepared as in Example 25, was poured into a polypropylene microtube. Subsequently, 88 mg of resin ECR8209F containing trypsin immobilized according to the above procedure was added to the microtube. Bulk polymerization was carried out at 10°C for 72 h. Afterward, the mixture was cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 3 to 37, with a peak maximum of 11, conversion was 95%, and regioselectivity (α) was 85%.

[0264] Example 33

[0265] Immobilization of trypsin

[0266] Trypsin was immobilized on polymer resin ECR8209F (Purilet) according to the procedure in Example 26, except that the resin was washed with a 0.5 M NaCl aqueous solution after enzyme immobilization. The enzyme activity was 0.665 U / g.

[0267] Ontological Aggregation of Lys-OEt

[0268] 200 µL of Lys-OEt, prepared as per Example 25, was poured into a polypropylene microtube. Subsequently, 111 mg of resin ECR8209F containing trypsin immobilized according to the above procedure was added to the microtube. Bulk polymerization was carried out at 10°C for 72 h. Afterward, the mixture was cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 3 to 42, with a peak maximum of 11, conversion was 88%, and regioselectivity (α) was 81%.

[0269] Example 34

[0270] Bulk polymerization of Lys-OEt is carried out simultaneously with in-situ desalination.

[0271] At 10°C, 200 mg of Lys-OEt dihydrochloride was thoroughly mixed with 150 µL of triethanolamine in a polypropylene microtube. Subsequently, 84 mg of agarose beads (purchased from Thermo Fisher Scientific) pre-washed five times with water and immobilized trypsin were added to the tube to initiate chemo-enzymatic polymerization. Bulk polymerization was carried out at 10°C for 72 h. The mixture was then cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 2 to 21, with a peak maximum of 5, conversion was 94%, and regioselectivity (α) was 88%.

[0272] Example 35

[0273] Bulk polymerization of Lys-OEt is carried out simultaneously with in-situ desalination.

[0274] At 10°C, 200 mg of Lys-OEt dihydrochloride was thoroughly mixed with 100 µL of triethanolamine and 50 µL of water in a polypropylene microtube. Subsequently, 84 mg of agarose beads (purchased from Thermo Fisher Scientific) pre-washed five times with water and immobilized trypsin were added to the tube to initiate chemo-enzymatic polymerization. Bulk polymerization was carried out at 10°C for 72 h. The mixture was then cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. DP ranged from 2 to 26, with a peak maximum of 7, conversion was 81%, and regioselectivity (α) was 93%.

[0275] Example 36

[0276] Bulk polymerization of Lys-OEt is carried out simultaneously with in-situ desalination.

[0277] According to Example 35, Lys-OEt was desalted and then polymerized in bulk, except that 100 µL of triethylamine was used as the organic base for desalting. The DP range was 2 to 11, with a peak maximum of 4, the conversion was 63%, and the regioselectivity (α) was 91%.

[0278] Example 37

[0279] Bulk polymerization of Lys-OEt is carried out simultaneously with in-situ desalination.

[0280] According to Example 35, Lys-OEt was desalted and then polymerized in bulk, except that 100 µL of tetramethylethylenediamine was used as the organic base for desalting. The DP range was 2 to 26, with a peak maximum of 4, the conversion was 44%, and the regioselectivity (α) was 100%.

[0281] Example 38

[0282] Arg-OEt bulk polymerization was carried out simultaneously with in-situ desalination.

[0283] At 10°C, 200 mg of arginine ethyl ester (Arg-OEt) dihydrochloride was thoroughly mixed with 100 µL of triethanolamine in a polypropylene microtube. Subsequently, 84 mg of agarose beads (purchased from Thermo Fisher Scientific) pre-washed five times with water and immobilized trypsin were added to the tube to initiate chemo-enzymatic polymerization. Bulk polymerization was carried out at 10°C for 168 h, with crude products collected at 24 h, 72 h, and 168 h. The final mixture was cooled to -30°C to stop the reaction. The filtered crude product was analyzed by MALDI-TOF MS and... 1 Characterized by 1H NMR. The results are summarized in Table 4.

[0284] Table 4

[0285]

[0286] Example 39

[0287] Arg-OEt bulk polymerization was carried out simultaneously with in-situ desalination.

[0288] Lys-OEt was desalted and then polymerized in bulk according to Example 38, except that the bulk polymerization was carried out at 30°C for 72 h. The DP range was 2 to 7, with a peak maximum of 3, the conversion was 29%, and the regioselectivity (α) was 100%.

[0289] Example 40

[0290] Arg-OEt bulk polymerization was carried out simultaneously with in-situ desalination.

[0291] Arg-OEt was desalted and then polymerized in bulk according to Example 38, except that the bulk polymerization was carried out at 60°C for 72 h. The DP was 2 to 7, with a peak maximum of 2, the conversion was 39%, and the regioselectivity (α) was 100%.

[0292] Example 41

[0293] Arg-OEt bulk polymerization was carried out simultaneously with in-situ desalination.

[0294] At 10°C, 200 mg of arginine ethyl ester (Arg-OEt) dihydrochloride was thoroughly mixed with 100 µL of triethylamine in a polypropylene microtube. Subsequently, 84 mg of agarose beads (purchased from Thermo Fisher Scientific) pre-washed five times with water and immobilized trypsin were added to the tube to initiate chemoenzymatic polymerization. Bulk polymerization was carried out at 10°C for 72 h. The DP range was 2 to 8, with a peak maximum of 3, the conversion was 56%, and the regioselectivity (α) was 100%.

[0295] Although the invention has been described herein with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the invention. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and apparatus of the invention without departing from the spirit and scope thereof. Therefore, the invention is intended to include modifications and variations within the scope of the appended claims and their equivalents.

[0296] Example 42

[0297] According to Example 10, Lys-OEt was desalted and then polymerized in bulk. Bulk polymerization was carried out at 10°C for 24 h, with crude samples taken out between 1 h and 24 h. The characterization results from the crude samples are available in Table 5.

[0298] Table 5. Parameters from the method in Example 42.

[0299]

[0300] Example 43

[0301] The method was performed for 72 h according to Example 42, except that 5 µl of acetone was added to the microtube for polymerization. The DP range was 3 to 29, with a peak maximum of 6, the conversion was 100%, and the regioselectivity (ε) was 95%.

[0302] Example 44

[0303] The method was performed according to Example 43, except that 20 µl of acetone was added to the microtube for polymerization. The DP range was 3 to 23, with a peak maximum of 6, the conversion was 100%, and the regioselectivity (ε) was 98%.

[0304] Example 45

[0305] The method was performed according to Example 43, except that 5 µl of acetonitrile was added to the microtube for polymerization. The DP range was 4 to 30, with a peak maximum of 6, the conversion was 100%, and the regioselectivity (ε) was 99%.

[0306] Example 46

[0307] The method was performed according to Example 43, except that 10 µl of acetonitrile was added to the microtube for polymerization. The DP range was 4 to 30, with a peak maximum of 6, the conversion was 100%, and the regioselectivity (ε) was 97%.

[0308] Example 47

[0309] The method was performed according to Example 43, except that 20 µl of acetonitrile was added to the microtube for polymerization. The DP range was 3 to 32, with a peak maximum of 5, the conversion was 100%, and the regioselectivity (ε) was 100%.

[0310] Example 48

[0311] The method was performed according to Example 43, except that 5 µl of ethanol was added to the microtube for polymerization. The DP range was 3 to 18, with a peak maximum of 5, the conversion was 100%, and the regioselectivity (ε) was 93%.

[0312] Example 49

[0313] The method was performed according to Example 43, except that 10 µl of ethanol was added to the microtube for polymerization. The DP range was 3 to 23, with a peak maximum of 6, the conversion was 100%, and the regioselectivity (ε) was 93%.

[0314] Example 50

[0315] The method was performed according to Example 43, except that 20 µl of ethanol was added to the microtube for polymerization. The DP range was 3 to 18, with a peak maximum of 6, the conversion was 100%, and the regioselectivity (ε) was 91%.

[0316] Example 51

[0317] The method was performed according to Example 43, except that 50 µl of ethanol was added to the microtube for polymerization. The DP range was 3 to 20, with a peak maximum of 6, the conversion was 100%, and the regioselectivity (ε) was 89%.

[0318] Example 52

[0319] The method was performed according to Example 43, except that 5 µl of glycerol was added to the microtube for polymerization. The DP range was 4 to 31, with a peak maximum of 6, the conversion was 50%, and the regioselectivity (ε) was 94%.

[0320] Example 53

[0321] The method was performed according to Example 43, except that 10 µl of glycerol was added to the microtube for polymerization. The DP range was 2 to 17, with a peak maximum of 5, the conversion was 40%, and the regioselectivity (ε) was 99%.

[0322] Example 54

[0323] Demineralization of Lys-OEt dihydrochloride:

[0324] 100 g of Amberlite IRA410 type II anion exchange resin (Cl form) (purchased from Sigma-Aldrich) was washed with 200 ml of 1 M NaOH aqueous solution to convert it to the OH form. The resin was further washed with 200 ml of deionized water, followed by 50 ml of ethanol. The resin was then added to 12.5 g of Lys-OEt dihydrochloride pre-suspended in 250 ml of chloroform and equilibrated at 4°C with gentle stirring for 60 min. The exchange resin was then filtered off, and the chloroform was evaporated at 10°C using a vacuum pump to produce liquid Lys-OEt. The recovered resin was regenerated by washing with 200 ml of 1 M NaOH aqueous solution and reused twice more to repeat the desalting procedure for the Lys-OEt dihydrochloride. A total of 23.9 g of liquid Lys-OEt was obtained.

[0325] Bulk polymerization of desalted Lys-OEt:

[0326] 16.9 g of Lys-OEt was poured into a round-bottom flask, along with 2.54 g of Novozym 435 (CALB immobilized on the polymer resin, purchased from Novozymes) and 1.69 ml of ethanol. Bulk polymerization was carried out at 10°C for 72 h with stirring. The crude product was characterized after filtration. The DP range was 2 to 25, with a peak maximum of 6, the conversion was 97%, and the regioselectivity (ε) was 85%.

[0327] Example 55

[0328] Demineralization of Lys-OEt dihydrochloride:

[0329] 360 g of Amberlite IRA410 type II anion exchange resin (Cl form) (purchased from Sigma-Aldrich) was washed with 360 ml of 1 M NaOH aqueous solution to convert it to the OH form. The resin was further washed with 360 ml of deionized water, followed by 90 ml of ethanol. The resin was then added to 45 g of Lys-OEt dihydrochloride pre-suspended in 900 ml of chloroform and equilibrated at 4°C with gentle stirring for 60 min. The exchange resin was then filtered off, and the chloroform was evaporated using a vacuum pump at 10°C to produce liquid Lys-OEt. The recovered resin was regenerated by washing with 360 ml of 1 M NaOH aqueous solution and reused three more times to repeat the desalting procedure for the Lys-OEt dihydrochloride. A total of 143 g of liquid Lys-OEt was obtained.

[0330] Bulk polymerization of desalted Lys-OEt:

[0331] 15 µl of Lys-OEt was poured into a DSC pan, 2.3 mg of Novozym 435 (CALB immobilized on the polymer resin, purchased from Novozymes) was added, and the pan was capped and sealed. Bulk polymerization was carried out at 10°C for 60 h. The DP range was 2 to 39, with a peak maximum of 7, and the regioselectivity (ε) was 94%.

[0332] Example 56

[0333] Demineralization of Lys-OEt dihydrochloride:

[0334] 100 g of Lys-OEt dihydrochloride was dissolved in 140 ml of 5 M NaOH aqueous solution in an ice bath. The resulting solution was mixed with 1 l of chloroform in a jacketed glass container at 10°C with vigorous stirring at 500 rpm for 15 min. The two phases were then separated without stirring for 15 min. The organic phase was collected and dried over anhydrous sodium sulfate. The chloroform was evaporated at 10°C using a vacuum pump to produce liquid Lys-OEt. The extraction was repeated twice more from the aqueous phase using fresh chloroform.

[0335] Bulk polymerization of desalted Lys-OEt:

[0336] 290 ml of Lys-OEt was poured into a jacketed glass reactor equipped with a Rushton impeller, and 43.5 g of Novozym 435 (CALB immobilized on the polymer resin, purchased from Novozymes) and 43.5 ml of ethanol were added. Bulk polymerization was carried out at 10°C for 72 h with stirring. The crude product was characterized after filtration. The DP range was 2 to 25, with a peak maximum of 5, the conversion was 85%, and the regioselectivity (ε) was 88%.

[0337] Example 57

[0338] Lys-OEt was desalted according to Example 10, and then polymerized in bulk in the presence of immobilized trypsin according to Example 22. Bulk polymerization was carried out at 10°C for 24 h, with crude samples taken out between 1 h and 24 h. The characterization results from the crude samples are available in Table 6.

[0339] Table 6. Parameters from the method in Example 57.

[0340]

[0341] Example 58

[0342] Trypsin fixation:

[0343] Trypsin was immobilized on polymer resin ECR8209F (Purilet) according to the procedure in Example 33, except that 15 g of ECR8209F resin was used. The immobilization yield was 8.4%. The enzyme activity was 0.447 U / g.

[0344] Bulk polymerization of desalted Lys-OEt:

[0345] 17.1 mL of Lys-OEt, desalted according to Example 25, was poured into a round-bottom flask. Subsequently, 14.2 g of resin ECR8209F containing trypsin fixed according to the above procedure was added to the flask. Bulk polymerization was carried out at 10°C for 96 h with stirring. The crude product was characterized after filtration. The DP range was 3 to 46, with a peak maximum of 10, the conversion was 78%, and the regioselectivity (α) was 88%.

[0346] Example 59

[0347] Lys-OEt dihydrochloride was desalted in situ according to Example 35. Subsequently, 71 mg of resin ECR8209F containing trypsin immobilized according to Example 58 was added to a microtube. Bulk polymerization was carried out at 10°C for 72 h. The crude product was characterized after filtration. The DP range was 2 to 29, with a peak maximum of 6, the conversion was 94%, and the regioselectivity (α) was 88%.

[0348] Example 60

[0349] Trypsin fixation:

[0350] Trypsin was immobilized on polymer resin ECR8209F (Purilet) according to the procedure in Example 26, except that after mixing and equilibrating the slurry, the resin was washed four times with 0.5 M NaCl aqueous solution, followed by two more washes with water. The immobilization yield was 68%. The enzyme activity was 0.928 U / g.

[0351] Recycling of immobilized trypsin:

[0352] The recyclability of the immobilized trypsin was tested. 200 µL of Lys-OEt, desalted according to Example 25, was poured into a polypropylene microtube. Subsequently, 160 mg of resin ECR8209F containing trypsin immobilized according to the above procedure was added to the tube. Bulk polymerization was carried out at 10°C for 72 h. After the initial polymerization, the product was washed with 1000 µL of water, centrifuged at 6000 rpm for 1 min, and the supernatant containing the resulting polylysine was removed. This washing step was repeated three times. Then, 200 µL of desalted Lys-OEt was added to begin the next polymerization cycle. Two repeated polymerization cycles were performed. The polymer from each cycle was characterized.

[0353] The results can be found in Table 7.

[0354] Table 7. Parameters from the method in Example 60.

[0355]

[0356] Example 61

[0357] Trypsin fixation:

[0358] 500 mg of polymeric resin ECR8209F (Purile Corporation) was washed with water at a resin:buffer ratio of 1:2 w / v, and excess liquid was filtered off. The washing was repeated four times. The washed resin was added to a solution of 25 mg / ml trypsin (from porcine pancreas, for biochemical and chemiluminescence companies) in 1X phosphate-buffered saline (pH 7.4) at a resin:buffer ratio of 1:4 w / v. The slurry was gently mixed for 18 h and then reequilibrated for 20 h without mixing. The supernatant was then collected, and the resin was washed ten times with 0.5 M NaCl aqueous solution, followed by two more washes with water. The fixed yield was 50%. The enzyme activity was 0.959 U / g.

[0359] Bulk polymerization of desalted Lys-OEt:

[0360] 200 µL of Lys-OEt, desalted according to Example 25, was poured into a polypropylene microtube. Subsequently, 130 mg of resin ECR8209F containing trypsin immobilized according to the above procedure was added to the tube. Bulk polymerization was carried out at 10°C for 72 h. The crude product was characterized after filtration. The DP range was 2 to 39, with a peak maximum of 12, the conversion was 97%, and the regioselectivity (α) was 92%.

[0361] Example 62

[0362] Trypsin fixation:

[0363] Trypsin was immobilized on polymer resin ECR8209F (Purilet) according to the procedure in Example 61, except that the resin:buffer ratio in the immobilization slurry was 1:10 w / v. The immobilization yield was 12%. The enzyme activity was 1.867 U / g.

[0364] Bulk polymerization of desalted Lys-OEt:

[0365] 200 µL of Lys-OEt, desalted according to Example 25, was poured into a polypropylene microtube. Subsequently, 77 mg of resin ECR8209F containing trypsin immobilized according to the above procedure was added to the tube. Bulk polymerization was carried out at 10°C for 72 h. The crude product was characterized after filtration. The DP range was 2 to 39, with a peak maximum of 12, the conversion was 87%, and the regioselectivity (α) was 78%.

[0366] Example 63

[0367] Trypsin fixation:

[0368] Trypsin was immobilized on polymer resin ECR8209F (Purilet) according to the procedure in Example 61, except that the concentration of trypsin in the immobilization buffer was 6.25 mg / ml. The immobilization yield was 63%. The enzyme activity was 0.824 U / g.

[0369] Bulk polymerization of desalted Lys-OEt:

[0370] 200 µL of Lys-OEt, desalted according to Example 25, was poured into a polypropylene microtube. Subsequently, 175 mg of resin ECR8209F containing trypsin immobilized according to the above procedure was added to the tube. Bulk polymerization was carried out at 10°C for 72 h. The crude product was characterized after filtration. The DP range was 2 to 39, with a peak maximum of 8, the conversion was 95%, and the regioselectivity (α) was 89%.

[0371] Example 64

[0372] 200 µL of Lys-OEt, desalted according to Example 25, was poured into a polypropylene microtube. Subsequently, 10 mg of natural trypsin (non-immobilized trypsin, derived from porcine pancreas, for biochemistry, and Hikari Co., Ltd.) was added to the tube. The activity of the natural trypsin was 4000 USP / mg. Bulk polymerization was carried out at 10°C for 72 h. The crude product was characterized after filtration. The DP range was 2 to 39, with a peak maximum of 9, the conversion was 85%, and the regioselectivity (α) was 80%.

[0373] Example 65

[0374] The method was performed according to Example 64, except that 20 mg of native trypsin (non-immobilized trypsin) was used for polymerization. The DP range was 2 to 39, with a peak maximum of 9, the conversion rate was 84%, and the regioselectivity (α) was 86%.

[0375] Example 66

[0376] Bulk polymerization of Arg-OEt is carried out simultaneously with in-situ desalination:

[0377] At 10°C, 200 mg of Arg-OEt dihydrochloride was thoroughly mixed with 100 µL of triethylamine and 20 µL of water in a polypropylene microtube. Subsequently, 10 mg of natural trypsin (non-immobilized trypsin 1:250, derived from porcine pancreas for cell culture, and manufactured by Hikari Corporation) was added to the tube to initiate chemically enzymatic polymerization. The activity of the natural trypsin was 920 USP / mg. Bulk polymerization was carried out at 10°C for 72 h. The crude product was characterized. DP ranged from 2 to 8, with a peak maximum of 3, indicating a conversion of 70% and a regioselectivity (α) of 100%.

[0378] Example 67

[0379] Bulk polymerization of Arg-OEt is carried out simultaneously with in-situ desalination:

[0380] Arg-OEt dihydrochloride was desalted in situ according to Example 66. Subsequently, 4 mg of native proteinase K (non-immobilized protease, recombinant, for biochemistry, from Fujifilm Wako) was added to the microtube. The activity of native proteinase K was 38 U / mg, where 1 U at 37°C utilized bovine blood-derived hemoglobin as a substrate to produce a peptide equivalent to 1 µmol of tyrosine (as a Folin chromogenic agent) per minute. Bulk polymerization was carried out at 10°C for 24 h. The crude product was characterized. DP was between 2 and 8, with a peak maximum of 4, the conversion rate was 71%, and the regioselectivity (α) was 100%.

[0381] Example 68

[0382] Bulk polymerization of Arg-OEt is carried out simultaneously with in-situ desalination:

[0383] The method was performed according to Example 67, except that the polymerization was carried out for 72 hours. DP ranged from 2 to 9, with a peak maximum of 4. Regioselectivity (α) was 100%.

[0384] Example 69

[0385] Bulk polymerization of Arg-OEt is carried out simultaneously with in-situ desalination:

[0386] Arg-OEt dihydrochloride was desalted in situ according to Example 66. Subsequently, 4 mg of natural α-chymotrypsin (from bovine pancreas, powder, TCI) was added to the microtube. The activity of natural α-chymotrypsin was 28 U / mg, where 1 U hydrolyzes 1.0 µmol of N-benzoyl-L-tyrosine ethyl ester per minute at 25°C and pH 7.8. Bulk polymerization was carried out at 10°C for 96 h, with crude samples collected between 6 h and 96 h. The characterization results from the crude samples are available in Table 8.

[0387] Table 8. Parameters from the method in Example 69.

[0388]

[0389] Example 70

[0390] Bulk polymerization of Arg-OEt is carried out simultaneously with in-situ desalination:

[0391] At 10°C, 200 mg of Arg-OEt dihydrochloride was thoroughly mixed with 50 µL of 5 M NaOH aqueous solution in a polypropylene microtube. Subsequently, 4 mg of natural α-chymotrypsin (the same as in Example 69) was added to the microtube. Bulk polymerization was carried out at 10°C for 72 h. The crude product was characterized. DP ranged from 2 to 21, with a peak maximum of 8, indicating a conversion of 81% and a regioselectivity (α) of 100%.

[0392] Polymer biodegradability

[0393] Biodegradability in the presence of wastewater sludge

[0394] Following the OECD 301F biodegradation protocol, the biodegradability of the freeze-dried polymer in the presence of activated sludge was tested. The results are shown in Table 9.

[0395] Table 9. Biodegradability under OECD 301F.

[0396]

[0397] Proteolytic degradation in the presence of trypsin

[0398] The degradability in the presence of trypsin was investigated in an in vitro proteolytic degradation assay. An aqueous solution of ε- or α-polylysine (7.5 mg) prepared according to Examples 42 and 57 in 0.5 mL of Milli-Q water was maintained at 30°C, and a solution of trypsin (1 mg) in 0.5 mL of Milli-Q water was added. The final solution was stirred for 12 h at 30°C and 1800 rpm using a ThermoMixer C (Eppendorf, Hamburg, Germany). As a control, the same reaction was performed in the absence of trypsin. After the reaction, trypsin was removed by ultrafiltration at 9000 rpm for 10 min using a Vivaspin™ 500 (MWCO: 10,000), and the filtrate was lyophilized. The molecular weight distribution of the resulting solids was analyzed by gel permeation chromatography (GPC) using a Nexera series (Shimadzu, Kyoto, Japan) with a system controller SCL-40. The sample (5 mg mL⁻¹) was eluted with 0.5 M AcOH and 0.1 M NaNO₃ aqueous solution at a flow rate of 1.0 mL min⁻¹ using a Shodex OHpak SB-803 HQ column (Resonac, Tokyo, Japan) at 40°C. The sample was analyzed using an RI detector RID-20A and a photodiode array detector SPD-M40 (Shimadzu Corporation). Chromatograms were analyzed using poly(ethylene glycol) standards via LabSolutions software (Shimadzu Corporation).

[0399] After trypsin treatment, the GPC chromatogram, including the peak of ε-polylysine, remained almost unchanged. In contrast, the GPC chromatogram of α-polylysine showed a significant peak shift towards lower molecular weights. This result clearly indicates that trypsin selectively cleaves the α-linked peptide bonds of polylysine, while the ε-linked peptide bonds are not recognized in the catalytic pocket of trypsin.

Claims

1. A method for preparing polylysine or polyarginine, the method comprising chemically enzymatically polymerizing a lysine ester in bulk in the presence of an immobilized or non-immobilized protease to regioselectively provide α-polylysine, or chemically enzymatically polymerizing a lysine ester in the presence of an immobilized or non-immobilized lipase to regioselectively provide ε-polylysine, or chemically enzymatically polymerizing an arginine ester in bulk in the presence of an immobilized or non-immobilized protease to regioselectively provide α-polyarginine.

2. The method according to claim 1, wherein, The lysine ester is selected from lysine alkyl esters, such as lysine C. 1-6 - The group consisting of alkyl esters and lysine aryl esters such as lysine phenyl esters and lysine benzyl esters, wherein the arginine ester is selected from arginine alkyl esters such as arginine C 1-6 - The group consisting of alkyl esters and arginine aryl esters, such as arginine phenyl ester and arginine benzyl ester.

3. The method according to claim 2, wherein, This lysine ester is selected from lysine C. 1-4 -alkyl esters, such as lysine methyl ester, lysine ethyl ester, lysine propyl ester, and lysine butyl ester, wherein the arginine ester is selected from arginine C. 1-4 -Alkyl esters, such as arginine methyl ester, arginine ethyl ester, arginine propyl ester and arginine butyl ester.

4. The method according to any one of the preceding claims, wherein, The protease is selected from the group consisting of: serine proteases such as trypsin, α-chymotrypsin, elastase, carboxypeptidase and subtilisin, and cysteine ​​proteases such as papain, fig protease, bromelain, annalin, calpain, caspase, papain, cathepsin, clostridium protease and actinidin.

5. The method according to any one of the preceding claims, wherein, This protease is selected from the group consisting of serine proteases such as trypsin and subtilisin, and cysteine ​​proteases such as papain.

6. The method according to any one of claims 1 to 3, wherein, This lipase is selected from lipase (EC3.1.1.3).

7. The method according to claim 6, wherein, The lipase was selected from the group consisting of: lipase from Candida antarctica B (i.e., Candida antarctica lipase B (CALB)), lipase from Aspergillus niger (i.e., Aspergillus niger lipase), lipase from Burkholderia cepacia (i.e., Burkholderia cepacia lipase), and lipase from Rhizopus oryzae (Rhizopus oryzae lipase).

8. The method according to any one of the preceding claims further comprises desalting a salt of the lysine ester, particularly an inorganic acid salt of the lysine ester, to provide the lysine ester, or desalting a salt of the arginine ester, particularly an inorganic acid salt of the arginine ester, to provide the arginine ester.

9. The method according to any one of the preceding claims, wherein, The chemical enzymatic polymerization is carried out at a temperature ranging from -20°C to 80°C, preferably from 0°C to 35°C.

10. The method according to any one of the preceding claims, wherein, The chemical enzymatic polymerization is carried out for a period of at least 12 hours, and particularly at least 20 hours.

11. The method according to any one of the preceding claims, wherein, The chemical enzymatic polymerization process can last up to 192 hours.

12. The method according to any one of the preceding claims, further comprising separating the immobilized enzyme or the non-immobilized enzyme from the polylysine or the polyarginine obtained by the chemical enzymatic polymerization.

13. The method of claim 12, further comprising applying the isolated immobilized enzyme or the non-immobilized enzyme to an additional chemically enzymatic polymerization of lysine esters or arginine esters.

14. The method according to any one of the preceding claims, wherein, The polylysine has a degree of polymerization in the range of 2 to 60, particularly 2 to 50, and the polyarginine has a degree of polymerization in the range of 2 to 30, particularly 2 to 20.

15. The method according to claim 14, wherein, The degree of polymerization of the polylysine has a peak maximum value in the range of 4 to 25, particularly 6 to 20, and the degree of polymerization of the polyarginine has a peak maximum value in the range of 2 to 5.

16. A method for controlling the regioselectivity of linear polylysine or polyarginine in the chemical enzymatic polymerization of lysine esters or arginine esters in bulk, wherein an immobilized or non-immobilized protease is used to regioselectively provide α-polylysine or α-polyarginine, or an immobilized or non-immobilized lipase is used to regioselectively provide ε-polylysine.

17. The method according to claim 16, wherein, The lysine ester is selected from lysine alkyl esters, such as lysine C. 1-6 - The group consisting of alkyl esters and lysine aryl esters such as lysine phenyl and lysine benzyl esters, wherein the arginine ester is selected from arginine alkyl esters such as arginine C 1-6 - The group consisting of alkyl esters and arginine aryl esters, such as arginine phenyl ester and arginine benzyl ester.

18. The method according to claim 17, wherein, This lysine ester is selected from lysine C. 1-4 -alkyl esters, such as lysine methyl ester, lysine ethyl ester, lysine propyl ester, and lysine butyl ester, wherein the arginine ester is selected from arginine C. 1-4 -Alkyl esters, such as arginine methyl ester, arginine ethyl ester, arginine propyl ester and arginine butyl ester.

19. The method according to any one of claims 16 to 18, wherein, The protease is selected from the group consisting of: serine proteases such as trypsin, α-chymotrypsin, elastase, carboxypeptidase and subtilisin, and cysteine ​​proteases such as papain, fig protease, bromelain, annalin, calpain, caspase, papain, cathepsin, clostridium protease and actinidin.

20. The method according to claim 19, wherein, This protease is selected from the group consisting of serine proteases such as trypsin and subtilisin, and cysteine ​​proteases such as papain.

21. The method according to any one of claims 16 to 18, wherein, This lipase is selected from lipase (EC3.1.1.3).

22. The method according to claim 21, wherein, The lipase was selected from the group consisting of: lipase from Candida antarctica B (i.e., Candida antarctica lipase B (CALB)), lipase from Aspergillus niger (i.e., Aspergillus niger lipase), lipase from Burkholderia cepacia (i.e., Burkholderia cepacia lipase), and lipase from Rhizopus oryzae (Rhizopus oryzae lipase).

23. The method according to any one of claims 16 to 22, wherein, The chemical enzymatic polymerization is carried out at a temperature ranging from -20°C to 80°C, preferably from 0°C to 35°C.

24. The method according to any one of claims 16 to 23, wherein, The chemical enzymatic polymerization is carried out for a period of at least 12 hours, particularly at least 20 hours, and preferably up to 192 hours.

25. The method according to any one of claims 16 to 24, wherein, The regioselectivity of α-polylysine is at least 60%, preferably at least 70%.

26. The method according to any one of claims 16 to 24, wherein, The regioselectivity of ε-polylysine can be at least 70%, preferably at least 80%.

27. The method according to any one of claims 16 to 24, wherein, The regioselectivity of ε-polyarginine can be at least 75%, preferably at least 90%.

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