WO2025243208A2

WO2025243208A2 - Process for the preparation and/or purification of polyethylene glycol and its derivatives, obtained product and uses thereof

Info

Publication number: WO2025243208A2
Application number: PCT/IB2025/055243
Authority: WO
Inventors: Claudia BENTO; Marianna KATZ; Nuno LOURENÇO TORRES; Carlos AFONSO
Original assignee: Hovione Farmaciencia SA
Current assignee: Hovione Farmaciencia SA
Priority date: 2024-05-20
Filing date: 2025-05-20
Publication date: 2025-11-27
Anticipated expiration: 2026-11-20

Abstract

The present disclosure relates to a novel process for the preparation and/or purification of polyethylene glycol (PEG) and its derivatives. More specifically, the disclosure concerns to methods that improve the purity, yield, and efficiency of PEG elongation/extension, as well as the obtained products and their several applications in industrial, pharmaceutical, and medical fields.

Description

D E S C R I P T I O N

PROCESS FOR THE PREPARATION AND/OR PURIFICATION OF POLYETHYLENE GLYCOL AND ITS DERIVATIVES, OBTAINED PRODUCT AND USES THEREOF

TECHNICAL FIELD

[0001] The present disclosure relates to a novel process for the preparation and/or purification of polyethylene glycol (PEG) and its derivatives. More specifically, the disclosure concerns methods that improve the purity, yield, and efficiency of PEG elongation/extension, as well as the obtained products and their several applications in industrial, pharmaceutical, and medical fields.

BACKGROUND

[0002] Poly(ethylene glycol) (PEG) is a synthetic, hydrophilic, and biocompatible polymer. Its chemical formula is H(OCH2CH2)_n OH where n corresponds to the number of units of ethylene oxide. PEGs are widely used in drug delivery due to their non-toxicity, low immunogenicity, and well-established safety profiles. The Food and Drug Administration (FDA) has selected PEG as the preferred choice for drug delivery systems compared to other polymers because of these key requisites.¹ PEGylation, a technique that covalently or noncovalently attaches PEG chains to small molecule drugs, peptides, and nucleic acids, has been shown to optimize the pharmacokinetics and pharmacodynamics of drugs, improving drug stability and reducing nonspecific protein absorption and macrophage uptake. ^2-5 This technique significantly prolongs circulation time due to the stealth effect of PEG. PEG has been chemically modified by introducing a variety of functional groups to synthesize tailored PEG derivatives, making this polymer even more suitable for clinical drug development. ^6,7 To date, over 30 PEGylated drugs have been approved for clinical applications, with a market size of over USD 10 billion.^8,9

[0003] The conventional process to synthesize PEG relies on anionic ring opening polymerization. This approach depends on a PEG initiator, usually a monomeric PEG molecule that is activated with a catalyst, which is typically potassium hydroxide. This PEG initiator is activated generating the alkoxide ion that will react with ethylene oxide resulting in the formation of a new ether bond. This process is repeated multiple times to form the polymer chain that is terminated when a quenching agent is added, such as acetic acid, to deactivate the catalyst and prevent further polymerization. ¹⁰¹¹ Due to the randomness of this conventional process to synthesize PEG, the products result in mixtures of many different molecules of varying length and molecular weight with disperse nature.¹² This hurdles the synthetic and purification process of PEGylated drugs, thereby compromising the reproductive quality. Although these mixtures are currently used for PEGylation of pharmaceuticals, they are not ideal since they suffer from limitations in maintaining consistent product composition across batches, have a challenging characterization, potential loss of pharmaceutical ingredient activity due to PEG size heterogeneity, and hurdles in obtaining FDA approval. ^{13 14} To address this issue, significant efforts have been made to synthesize monodisperse PEGs via stepwise organic synthesis with a narrow molecular weight and a polydispersity index (PDI) of 1.0. However, these new methods usually involve step-by-step iterations which have some drawbacks such as low reaction rates and the need for chromatographic purification after every step. ¹⁵

[0004] The attempt to synthesize monodisperse PEGs dates to 1936 when Fordyce first reported PEG synthesis by reacting dichloroethane with ethylene glycol alkoxides. Even though he was able to elongate PEG chain, a mixture of different length PEGs was obtained.

[0005] Over the years, various methods have been attempted to extend the chain of PEGs. In 1987, a tosylation approach was utilized, but it resulted in a mixture of PEGs with varying lengths. ¹⁶ To address this challenge, a new method was reported in 1992 involving the extension of a PEG with 54 ethylene oxide units using tosyl chloride synthesis. This method incorporated an additional step of monoprotecting the PEG chain with a trityl group to enhance reaction control and prevent any side reactions. ¹⁷

[0006] In 2001, Zada et al. first reported the use of benzyl to monoprotect PEG (Scheme 1) They observed that by blocking one end group of PEG with a protecting group, such as benzyl, which can be removed at a later stage in the synthesis, will not lead to the formation of a large mixture of oligomers. Moreover, they concluded that monoprotection with benzyl as protecting group and NaH as base improved the yields and that by using more sophisticated separation and purification methods, such as preparative size-exclusion chromatography improved the final purity. ¹⁸

Scheme 1. Use of benzyl to monoprotect PEG. [0007] Gothatd et al. developed a synthetic approach to synthesize PEG 6, 10 and 12 without the need for column chromatographic separation (Scheme 2). To do so, a bidirectional growth of the PEG chains was made using easy-to-remove trityl groups that are easily removed in liquid-liquid extraction. However, this method had faults in higher molecular weight polymers.¹⁹ To overcome impurities and further issues with monosubstitution, a novel homostar approach was exploited by using a hub derived from l,3,5-tris-(bromomethyl)benzene that was linked to each PEG chain through a benzyl ether that works as protecting group. The chain grown unidirectionally by attaching building blocks into each end of existing chain and the branched structure facilitated the chromatographic purification of oligomeric intermediates. Moreover, even though with this method, chromatography was used to isolate products, it was noted that the large size and higher polarity of the homostar provided potential for purification by alternative size-discriminating techniques such as organic solvent nano-filtration. ²⁰

Scheme 2. Synthetic approach to synthesize PEG 6, 10 and 12 without the need for column chromatographic separation. [0008] The approach of macrocyclization of PEGs (Scheme 3) was used with sulphates that undergo iterative nucleophilic ring-opening reactions with a mononucleophile. This approach has the advantage of avoiding the iterative protection and activation of the -OH terminal PEG, thereby minimizing the synthetic steps. The nucleophilic attack allowed for the easy monofunctionalization of PEG, leading to the largest monodispersed monomethoxy-PEGs synthesized with 64 EO (ethylene oxide) units. However, a disadvantage of this method is that all the intermediates were purified by flash chromatography. ^21,22

Scheme 3. Macrocyclization of PEGs.

[0009] To avoid the chromatographic separation, Wawro et al. developed a chromatography-free synthetic method of monodisperse PEGs, with high purity of the product. The protection was made using triphenylmethyl as protecting group resulting in a mixture of mono and bisprotected PEG. The mixture was then reacted with tosyl chloride, resulting in a higher mixture containing monoprotected PEG tosylated and bisprotected PEG. Afterwards, a reaction is made with this mixture with a free PEG that will only react with the tosylated PEG. In the end, the protecting group is removed obtaining free PEG and monotosylated PEG that easily portioned between organic and aqueous phases. ²³ However, there were limitations with this method. The longer the PEG chain, this tosylated PEG will no longer go to the organic phase. Moreover, there is a decreasing purity of longer oligomers since within each chain extension cycle more contaminations were observed in the final product.

[0010] Solid phase technology was also applied for the synthesis of monodisperse PEGs to attempt to avoid the chromatographic separations and Khanal et al. attempted to overcome this issue. By employing solid phase chemistry to do the monodisperse PEG synthesis, they were able to avoid the tedious purifications required for this procedure. The purification of intermediates and product was made by washing and the used excess of reactants allowed high conversions overcoming the low efficiency of the Williamson ether reaction. The pure products were obtained without chromatography until PEG12 length. However, when they attempted to move forward to higher PEG lengths, they found many challenges as they went through the synthesis of PEG16 and PEG20: some chains did not react further therefore it became difficult to remove shorter PEGs from longer PEGs. ²⁴

[0011] It is often difficult or impossible to prepare heterobifunctional PEGs with high purity using existing methods since the bis-substituted PEG is inevitably formed, leading to the need of chromatography to separate this mixture. Recently, in CN111936549A, an approach was developed to overcome the high impurity level from the step of monosubstitution. It consists of the substitution of PEG monomer with trityl as protecting group that will form monosubstituted PEG and bis-substituted PEG. To isolate the monosubstituted step, a carboxylic acid is introduced by the esterification of the free a hydroxyl group of monodisperse PEG containing one terminal trityl group to increase hydrophilicity thereby enabling extraction and removal of ditritylated impurities to an organic layer.²⁵ In JP2008174755A, two different protecting groups are used, allowing the chain extension without the bissubstituted PEG complicating the following chain extension steps. ²⁶

[0012] EP1594440B1 described the reaction of bistosylated PEG monomer with monosubstituted PEG elongating the chain. This was an effective approach to extend the chain but the monosubstitution step required chromatographic isolation to remove the bis substituted impurity.²⁷ The same approach is followed on CN104892372B where the chain extension is made with a leaving group approach and the monosubstitution is carried out with Dihydropyran. Even though they performed an efficient chain extension yielding uniform PEGs, it was required a chromatographic isolation of the monosubstituted PEG. ²⁸

[0013] Traditional methods for producing uniform PEGs encounter challenges during a monosubstitution step where unwanted impurities must be removed with chromatography separation. To create a more efficient process for large-scale production, it is necessary to improve this monosubstitution step by reducing impurities avoiding the extra purification steps.

[0014] Purification is a critical stage in various industrial processes, but certain chemical mixtures can be difficult to isolate, which may result in reduced productivity and efficiency. Some of the challenging techniques include crystallization, distillation, and gas or liquid-liquid extractions. Liquid-liquid extractions are used in many industries to separate components from a mixture based on their solubility in two immiscible liquids. However, traditional solvents have drawbacks such as toxicity, volatility, and environmental impact. To overcome these limitations, researchers have been exploring new solvents. To this end, deep eutectic solvents (DES) have gained attention in recent years owing to their unique properties such as high biodegradability, low toxicity, high thermal stability, low volatility, nonflammability, high air stability, bio-renewability, and biocompatibility. DES are formed by combining two or more solid compounds, typically salts, with a lower melting point than each individual component. This unique characteristic allows eutectic solvents to remain in a liquid state at ambient temperatures, providing a versatile and environmentally friendly alternative to conventional organic solvents. These solvents are viewed as promising substitutes for conventional solvents, as they are more eco-friendly, typically less costly, easy to prepare, and increasingly used in industrial processes to purify and separate products during chemical reactions. DES are a subclass of ionic liquids with unique characteristics. These are formed by mixing different components, which form a eutectic mixture with a much lower melting point than any of the individual components. The first generation of these solvents were created by mixing quaternary ammonium salts with hydrogen donors, such as amines and carboxylic acids. A DES was first discovered in 2003, which was a mixture of choline chloride and urea in a 1:2 mole ratio, respectively. Choline chloride has a melting point of 302°C, while urea's is 133°C. However, the eutectic mixture melts at 12°C.³²

[0015] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

GENERAL DESCRIPTION

[0016] The present disclosure relates to an improved process for the elongation and/or purification of polyethylene glycol (PEG) and its derivatives. The novel process enhances the purity, yield, and efficiency of PEG elongation. Additionally, the disclosure describes the characteristics of the obtained PEG products.

[0017] The present disclosure relates to a process for the discrete PEG and its derivatives synthesis that combines innovative biological and chemical techniques. This method relies on the use of a biocatalyst to produce monosubstituted PEG with minimal impurity content, overcoming the limitations of existing methods, and delivering a monosubstituted PEG with significantly lower levels of impurity. After this step, the chain extension is then enabled using leaving groups. This ensures that the elongated polymer remains monodisperse. Additionally, the final conditions used to remove the protective groups are relatively milder compared to other reported methods.

[0018] The process of the present disclosure enables the production of polyethylene glycol on an industrial scale with high reproducibility and improved sustainability. The method is designed to be robust and scalable, ensuring consistent quality and performance across multiple production batches.

[0019] The polyethylene glycol (PEG) obtained by the method disclosed herein is characterized by being non-toxic, non-immunogenic, and chemically stable.

[0020] Furthermore, the process yields PEG with an improved polydispersity index, indicating a narrower molecular weight distribution, with batch-to batch consistency. The polydispersity index of the PEG obtained with the method of the present disclosure, prior to any purification step, is about 1.00; preferably ranging from 1.00-1.10. [0021] Polyethylene glycol (PEG) obtained through methods described in the prior art is associated with several disadvantages that limit its applicability in high-purity and high-performance formulations. In particular, such conventional methods often result in PEG products exhibiting a higher polydispersity index, usually above 1.10, indicative of a broader molecular weight distribution and reduced uniformity. Moreover, the PEG produced by these methods typically displays lower purity levels, necessitating additional purification steps, and a reduced shelf life, which compromises its long-term stability and usability, especially in pharmaceutical and biomedical applications.

[0022] The polyethylene glycol of the present disclosure, characterized by its exceptional purity, nontoxicity, non-immunogenicity, and chemical stability, and further distinguished by a remarkably low polydispersity index of not more than 1.10, may be used in the pharmaceutical and medical device industries.

[0023] In pharmaceutical formulations, the PEG may serve as an excipient, solubilizer, or stabilizing agent for small molecules, peptides, and biologies, including monoclonal antibodies, owing to its uniform molecular weight distribution and absence of toxic or immunogenic impurities. It is especially advantageous for use in drug delivery systems, such as PEGylated therapeutic agents, where the reduced heterogeneity minimizes immunological responses and enhances pharmacokinetic profiles.

[0024] In the medical devices field, the polyethylene glycol of the present disclosure may be used in coatings for implantable devices, catheters, and biosensors, where its chemical stability and biocompatibility reduce the risk of biofouling and inflammatory responses, thereby prolonging device functionality and patient safety. The high degree of uniformity and stability of the polyethylene glycol also make it suitable for use in hydrogel systems, wound dressings, and tissue engineering scaffolds requiring predictable degradation profiles and minimal leachable.

[0025] An aspect of the present disclosure relates to a method for obtaining an extended high molecular weight monodispersed polyethylene glycol of general formula (I'") formula (I'") comprising a step of enzymatic mono esterification of a polyethylene glycol of general formula (II) with a carboxylic acid or carboxylic acid derivative of general formula (III) formula (II) formula (III) to obtain a mono esterified polyethylene glycol of general formula (IV), formula (IV) wherein: the enzymatic mono esterification is performed in a suitable organic solvent and catalysed by a first enzyme selected from the group consisting of: lipase, esterase, protease, transferase, ligase or mixtures thereof;

Ri and R2 and independently selected;

Ri is an aliphatic or aromatic amine or a substituted or non-substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

R2 is hydrogen, a cycloalkyl or a C1-C12 substituted or non-substituted alkyl, alkenyl, or alkynyl; n is at least 1; p is 0 or at least 1.

[0026] An aspect of the present disclosure relates to a method for obtaining an extended high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I'): formula (I) formula (I') comprising a step of enzymatic mono esterification, using a first enzyme in a first suitable organic solvent of a polyethylene glycol of general formula (II) with a carboxylic acid or carboxylic acid derivative of general formula (III), formula (II) formula (III) to obtain a mono esterified polyethylene glycol of general formula (IV), formula (IV) reacting the compound of general formula (IV) with a leaving group (LGi), in a second suitable organic solvent, to obtain the compound of general formula (V) formula (V) reacting the compound of general formula (IV) or a compound of general formula (IV') formula (IV') with the compound of general formula (V), in a third suitable organic solvent and in the presence of a first base, to obtain the compound of general formula (VI) or the compound of general formula

VI', respectively, formula (VI') formula (VI) formula (VI') reacting the compound of general formula (VI) or the compound of general formula (VI'), with a second enzyme, in a suitable first aqueous-based or first water miscible solvent, to obtain the polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I'), respectively, wherein: the first enzyme and the second enzyme are independently selected from the group consisting of: lipase, esterase, protease, transferase, ligase or mixtures thereof;

Ri, Ri-, and R2 and independently selected;

Ri and Ri- are selected from the group consisting of: aliphatic amine, aromatic amine, substituted or non-substituted alkyl, substituted or non-substituted alkenyl, substituted or non-substituted alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;

R2 is selected from the group consisting of: hydrogen, cycloalkyl, C1-C12 substituted or nonsubstituted alkyl, C1-C12 substituted or non-substituted alkenyl, or C1-C12 substituted or nonsubstituted alkynyl; n and n' are independently selected; n and n' are at least 1. [0027] The present disclosure provides a safer alternative for PEG synthesis and eliminates also the need for high time consuming and costly purification techniques such as column chromatography, which are typically employed to address impurity issues from the monosubstitution step, thereby promoting the adoption of green chemistry practices.

[0028] It was surprisingly found that specific enzymes, selected from lipase, esterase, protease, transferase, ligase or mixtures thereof, allow the enzymatic mono esterification of polyethylene glycol, allowing to obtain mono esterified PEG'S through esterification in high yields and high selectivity without needing to perform purification by column chromatography.

[0029] In was also surprisingly found that the method of the present disclosure provides improved results even at or above atmosphere pressure (usually a pressure equal or above 101.32500 kPa) (1 atmosphere).

[0030] Polyethylene glycol is a polymer that is widely used due to its versatility, safety, and biocompatibility. It plays an important role in PEGylation, which involves attaching PEG chains for example but not limited to therapeutic molecules and has transformed drug delivery. PEGylation improves drug solubility, extends circulation time by reducing renal clearance, and reduces immunogenicity, thereby improving the effectiveness and safety of pharmaceuticals. The present disclosure also describes the valuable application of these novel compounds in the fields of diagnostics and therapeutics, among others.

[0031] In an embodiment for better results, the molecular weight of the high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') is at least 50 g/mol; preferably ranging from 150 g/mol - 20000 g/mol; more preferably 195 g/mol - 10000 g/mol.

[0032] In an embodiment for better results, the molecular weight of the high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') ranges from 50 g/mol - 30000 g/mol; preferably 150 g/mol - 20000 g/mol; more preferably 195 g/mol - 10000 g/mol.

[0033] In an embodiment for better results, 2n or n+n' ranges from 2-200; preferably 5-100; more preferably 8-60.

[0034] In an embodiment for better results, n or n' ranges from 1-100; preferably 2-50; more preferably 4-30.

[0035] In an embodiment for better results, the concentration of the polyethylene glycol of general formula (II) in the first suitable organic solvent ranges from 0.005 mol/L to 50 mol/L; preferably 0.5 to 5 mol/L; more preferably 1 mol/L.

[0036] In an embodiment for better results, the step of reacting the compound of general formula (IV) with a leaving group (LGi) is performed at a temperature ranging from 25-100°C; preferably 25-50 °C. [0037] In an embodiment for better results, the step of reacting the compound of general formula (IV) or a compound of general formula (IV') with the compound of general formula (V) is performed at a temperature ranging from 24-120°C; preferably 30-50 °C.

[0038] In an embodiment for better results, LGi is selected from p-toluenesulfonate or methanesulfonate; preferably p-toluenesulfonate.

[0039] In an embodiment for better results, the step of reacting the compound of general formula (VI) or the compound of general formula (VI'), with a second enzyme, is performed at a temperature ranging from 5-100°C; preferably 25-50 °C.

[0040] In an embodiment for better results, R2 is selected from CH3, CH2CH3, CH2CH2CH3 or hydrogen; preferably CH3, CH2CH3 or hydrogen; more preferably CH3 or hydrogen.

[0041] In an embodiment for better results, R2 is hydrogen.

[0042] In an embodiment for better results, Ri and/or Ri- is a non-substituted alkyl, a non-substituted alkenyl, a non-substituted alkynyl, a non-substituted cycloalkyl, a non-substituted heterocycloalkyl, a non-substituted aryl or a non-substituted heteroaryl.

[0043] In an embodiment for better results, Ri and/or Ri- is a substituted or non-substituted phenyl, a substituted or non-substituted naphthalenyl, a substituted or non-substituted tetrahydronephthalenyl or a substituted or non-substituted indane; preferably a non-substituted phenyl, a non-substituted naphthalenyl, a non-substituted tetrahydronephthalenyl or a non-substituted indane.

[0044] In an embodiment for better results, Ri and/or Ri- is a substituted or non-substituted monocyclic aromatic ring selected from the list consisting of: furyl, furazanyl, imidazolyl, isothiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyranyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, pyridyl, thienyl, thiazolyl, triazolyl, tetrazolyl, triazinyl, tetrazinyl; preferably a non-substituted monocyclic aromatic ring selected from the list consisting of: furyl, furazanyl, imidazolyl, isothiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyranyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, pyridyl, thienyl, thiazolyl, triazolyl, tetrazolyl, triazinyl, tetrazinyl.

[0045] In an embodiment for better results, Ri and/or Ri- is a substituted or non-substituted bicyclic aromatic ring selected from the group consisting of: azaindolyl, benzothienyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzoxadiazolyl, benzothiadiazolyl, benzofuranyl, cinnolinyl, furopyridyl, imidazopyridyl, indolyl, isoindolyl, isobenzofuranyl, indolizinyl, indazolyl, isoquinolinyl, naphthyridinyl, quinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pteridinyl, purinyl, pyrrolopyridyl; preferably Ri is a non-substituted bicyclic aromatic ring selected from the group consisting of: azaindolyl, benzothienyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzoxadiazolyl, benzothiadiazolyl, benzofuranyl, cinnolinyl, furopyridyl, imidazopyridyl, indolyl, isoindolyl, isobenzofuranyl, indolizinyl, indazolyl, isoquinolinyl, naphthyridinyl, quinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pteridinyl, purinyl, pyrrolopyridyl.

[0046] In an embodiment for better results, the compound of formula IV or formula IV' wherein the compound is selected from the list consisting of:

(Benzoyl) polyethylene glycol; (3-Aminophenyl)acetate polyethylene glycol; (4- Aminophenyl)acetate polyethylene glycol; (2-Aminophenyl)acetate polyethylene glycol; (3- Nitrophenyl)acetate polyethylene glycol; (4-Nitrophenyl)acetate polyethylene glycol; (2- Nitrophenyl)acetate polyethylene glycol; (3-Nitrobenzoyl) polyethylene glycol; (4-Nitrobenzoyl) polyethylene glycol; (2-Nitrobenzoyl) polyethylene glycol; (3-Methoxyphenyl)acetate polyethylene glycol; (4-Methoxyphenyl)acetate polyethylene glycol; (2-Methoxyphenyl)acetate polyethylene glycol; (3-Methylphenyl)acetate polyethylene glycol; (4-Methylphenyl)acetate polyethylene glycol; (2-Methylphenyl)acetate polyethylene glycol; (3-Methoxycinnamoyl) polyethylene glycol; (2- Tiophen-3-yl)acetate polyethylene glycol; ((E)-5-Methoxypent-4-enoate) polyethylene glycol; (2- Furan-3-yl)acetate polyethylene glycol; (2-Quinolin-3-yl)acetate polyethylene glycol; (2-(3- Methoxyphenyl)-2-oxoacetyl) polyethylene glycol; (3-Butynoyl) polyethylene glycol; (Maleimidylacetyl) polyethylene glycol; (Pyridin-3-yl)acetate polyethylene glycol; (Pyridin-4- yl)acetate polyethylene glycol; (Maleimidylpropionyl) polyethylene glycol; (Maleoyl) polyethylene glycol; (3-Acetamidophenylacetyl) polyethylene glycol; (3-Methoxycinnamoyl) polyethylene glycol; (Methyl-(3-Methoxy)cinnamoyl) polyethylene glycol; (Triphenylacetyl) polyethylene glycol; (Diphenylacetyl) polyethylene glycol; (3-Hydroxyphenyl)acetate polyethylene glycol; (4- Hydroxyphenyl)acetate polyethylene glycol; or (2-Hydroxyphenyl)acetate polyethylene glycol.

[0047] Based on the International Union of Pure and Applied Chemistry (IUPAC) definitions, an alkyl group is defined as a univalent group derived from alkanes by removal of a hydrogen atom from any carbon atom -Cnbhn+i. The groups derived by removal of a hydrogen atom from a terminal carbon atom of unbranched alkanes form a subclass of normal alkyl (n-alkyl) groups H (CF Jn. The groups RCH2, R2CH (R * H), and R3C (R * H) are primary, secondary and tertiary alkyl groups, respectively.

[0048] "Alkyl" includes "lower alkyl" and extends to cover carbon fragments having up to 30 carbon atoms. Examples of alkyl groups include octyl, nonyl, norbornyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, eicosyl, 3,7-diethyl-2,2-dimethyl-4 -propylnonyl, 2-(cyclododecyl)ethyl, adamantyl, and the like.

[0049] "Lower alkyl" means alkyl groups of from 1 to 7 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 2-methylcyclopropyl, cyclopropylmethyl, and the like.

[0050] Alkyl, alkenyl and alkynyl chain comprises branched and unbranched chains, substituted or nonsubstituted. [0051] As used herein, the term "optionally substituted" typically refers to from zero to four substituents, wherein the substituents are each independently selected. Each of the independently selected substituents may be the same or different than other substituents. For example, the substituents of an R group of a formula may be optionally substituted (e.g., from 1 to 4 times) with independently selected H, halogen, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain and amino acid.

[0052] In the present disclosure 'C1-C12 alkyl' refers to a linear or branched saturated hydrocarbon group containing from one to twelve carbon atoms. Examples of 'C1-C12 alkyl' include methyl, ethyl, isopropyl, n-propyl, tert-butyl, sec-butyl, n-butyl, isobutyl, n-pentyl, isopentyl, n-hexyl. Preferably the hydrocarbon is linear.

[0053] In the present disclosure 'Halogen' as used herein refers to a fluoride (F), chlorine (Cl), bromide (Br) or iodide (I), unless otherwise specified.

[0054] In the present disclosure 'Ci-Cg Haloalkyl' as used herein refers to a Ci-Cg alkyl group as defined above substituted by one or more halogen atoms.

[0055] In the present disclosure 'Aryl' as used herein refers to a Cg-C monocyclic or bicyclic hydrocarbon ring wherein at least one ring is aromatic. Examples of 'Aryl' group include phenyl, naphthalenyl, tetrahydronephthalenyl and indane.

[0056] In the present disclosure 'Ci-Cg alkoxy' as used herein refers to a -O(Ci-Cg alkyl) group wherein Ci-Cg alkyl is as defined above. Examples of such groups include methoxy, ethoxy, isopropoxy, butoxy, pentoxy and hexyloxy.

[0057] In the present disclosure 'Ci-Cg haloalkoxy' as used herein refers to a -O(Ci-Cg alkyl) group as defined above substituted by one or more halogen atoms.

[0058] In the present disclosure 'Heteroaryl' as used herein refers to a 5-6 membered monocyclic aromatic or a fused 8-10 membered bicyclic aromatic ring, which might be partially saturated, which monocyclic or bicyclic ring contains 1 to 4 heteroatoms selected from oxygen, nitrogen, and sulphur. Examples of monocyclic aromatic ring include furyl, furazanyl, imidazolyl, isothiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyranyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, pyridyl, thienyl, thiazolyl, triazolyl, tetrazolyl, triazinyl, tetrazinyl and the like. Examples of such bicyclic aromatic rings include azaindolyl, benzothienyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzoxadiazolyl, benzothiadiazolyl, benzofuranyl, cinnolinyl, furopyridyl, imidazopyridyl, indolyl, isoindolyl, isobenzofuranyl, indolizinyl, indazolyl, isoquinolinyl, naphthyridinyl, quinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pteridinyl, purinyl, pyrrolopyridyl. [0059] In the present disclosure 'Bicyclic ring' and 'Fused' in the context of a bicyclic ring refers to two rings which are joined together across a bond between two atoms (e.g. naphthalene), across a sequence of atoms to form a bridge (e.g. quinuclidine, adamantyl) or together at a single atom to form a spiro compound (e.g. l,4-dioxa-8-aza-spiro[4.5]decane).

[0060] In the present disclosure 'Substituted aryl' or 'Substituted heteroaryl' used herein with reference to R group means that a particular R group (e.g. Ra, Rb, Rc) can be substituted with one or more groups selected from Rx, halogen, OH, ORx, SH, SRx, OCORx, SCORx, COOH, NH2, NO2, CN, NHRx, NRxRy, CORx, CSRx, COORx, OPhRxRy, CONH₂, CONHRx, CONxNy, CONHOH, CONHNH2, CONHORx, CH2CH2NRXRX, CH2CH₂NRxRy, NHCONH2, NRxCORy, NHCORx, CONHPhRx, CONRxRx, CONHNH2, COHRx, NHCORx, NHSO2RX, wherein Rx and Ry are independently selected from Ci-Cg alkyl, Ci-Cg-alkoxy, Ci-Cg haloalkyl, Ci-Cg haloalkoxy, aryl, heteroaryl, C3-C10 cycloalkyl, alkyl Ci-Cg aryl and heterocyclyl, or Rx and Ry, together with the heteroatom to which they are joined, can form an heterocyclyl.

[0061] In the present disclosure 'Unsubstituted heteroaryl' as used herein refers to pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-5-yl, furan-2-yl or indolyl.

[0062] As used herein the term "aryl" refers to an optionally substituted mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, benzyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. "Optionally substituted aryl" includes aryl compounds having from zero to four substituents, and "substituted aryl" includes aryl compounds having one or more substituents.

[0063] The term "bicyclic" represents either an unsaturated or saturated stable 7- to 12- membered bridged or fused bicyclic carbon ring. The bicyclic ring may be attached at any carbon atom which affords a stable structure. The term includes, but is not limited to, naphthyl, dicyclohexyl, dicyclohexenyl, and the like.

[0064] As used herein, the term "halogen" or "halo" includes bromide, chlorine, fluoride, and iodide.

[0065] The term "haloalkyl" as used herein refers to an alkyl radical bearing at least one halogen substituent, for example, chloromethyl, fluoroethyl or trifluoromethyl and the like.

[0066] In an embodiment for better results, the carboxylic acid or carboxylic acid derivative is an aryl or alkyl-based carboxylic acid-containing molecule, optionally comprising one or various substitutions selected from alkyl, amino, nitro, methoxy, methyl, hydroxyl, and/or halogens.

[0067] In an embodiment for better results, the carboxylic acid or carboxylic acid derivative is selected from the list consisting of the following TablelA-lB (Table 1): [0068] Table 1A: Carboxylic acid or carboxylic acid derivatives.

[0069] Table IB: Carboxylic acid or carboxylic acid derivatives.

[0070] In an embodiment for better results, the carboxylic acid or carboxylic acid derivative is:

[0071] In an embodiment for better results, the carboxylic acid or carboxylic acid derivative is selected from the group consisting of: benzoic acid, amino-substituted phenylacetic acid, nitro-substituted phenylacetic acid, nitro-substituted benzoic acid, methoxy-substituted phenylacetic acid, methylsubstituted phenylacetic acid, 3-methoxycinnamic acid, 2-(tiophen-3-yl)acetic acid, (E)-5-methoxypent- 4-enoic acid, 2-(furan-3-yl)acetic acid, 2-(quinolin-3-yl)acetic acid, 2-(3-methoxyphenyl)-2-oxoacetic acid, 3-butynoic acid, maleimidyl acetic acid, pyridin-3-yl acetic acid, pyridin-4-yl acetic acid, maleimidyl propionic acid, maleic anhydride, 3-acetamidophenylacetic acid, 3-methoxycinnamic acid, methyl-(3- methox)cinnamate, triphenyl acetic acid, diphenyl acetic acid, hydroxyl-substituted phenylacetic acid, phenyl acetic acid, (3-methoxy) phenylacetate, triglycerides, diglycerides, monoglycerides, glucuronic acid, isosaccharinic acid, gluconic acid or glucaric acid, or esters thereof.

[0072] In an embodiment for better results, the carboxylic acid or carboxylic acid derivative is selected from the list consisting of: 3-aminophenylacetic acid, 4-aminophenylacetic acid, 2-aminophenylacetic acid, 3-nitrophenylacetic acid, 4-nitrophenylacetic acid, 2-nitrophenylacetic acid, 3- nitrobenzoic acid, 4- nitrobenzoic acid, 2-nitrobenzoic acid, 3-methoxyphenylacetic acid, 4-methoxyphenylacetic acid, 2- methoxyphenylacetic acid, 3-methylphenylacetic acid, 4-methylphenylacetic acid, 2-methylphenylacetic acid, 3-methoxycinnamic acid, 2-(tiophen-3-yl)acetic acid, (E)-5-methoxypent-4-enoic acid, 2-(furan-3- yl)acetic acid, 2-(quinolin-3-yl)acetic acid, 2-(3-methoxyphenyl)-2-oxoacetic acid, 3-butynoic acid, maleimidyl acetic acid, pyridin-3-yl acetic acid, pyridin-4-yl acetic acid, maleimidyl propionic acid, maleic anhydride, 3-acetamidophenylacetic acid, 3-methoxycinnamic acid, methyl-(3-methoxy)cinnamate, triphenyl acetic acid, diphenyl acetic acid, 3-hydroxy phenylacetic acid, 4-hydroxy phenylacetic acid, 2- hydroxy phenylacetic acid, phenyl acetic acid, (3-methoxy) phenylacetate, glucuronic acid, isosaccharinic acid, gluconic acid or glucaric acid, or esters thereof.

[0073] In an embodiment for better results, the carboxylic acid or carboxylic acid derivative is selected from the list consisting of: 2-(m-tolyl)acetic acid, 3-methoxyphenylacetic acid, (3-methoxy) phenylacetate, methyl-3-methoxy cinnamate, glucuronic acid and maleimidyl acetic acid; preferably 3- methoxyphenyl acetic acid or (3-methoxy) phenylacetate.

[0074] In an embodiment for better results, the carboxylic acid or carboxylic acid derivative is selected from the group consisting of: alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, ornithine, citrulline, diaminobutyric acid, p-alanine, y-aminobutyric acid, hydroxylysine, hydroxyproline, desmosine, or isodesmosine, or esters thereof.

[0075] In an embodiment for better results, the carboxylic acid or carboxylic acid derivative is selected from the group consisting of: 3-methyoxyphenylacetic acid; 3-methylphenylacetic acid; phenylacetic acid; 3-aminophenylacetic acid; 4-aminophenylacetic acid; 3-hydroxyphenylacetic acid; Benzoic acid; 3- acetaminophenylacetic acid; 4-nitrophenylacetic acid; Triphenylacetic acid; 2-methoxyphenylacetic acid, or esters thereof.

[0076] In an embodiment for better results, p is 1.

[0077] In an embodiment for better results, the amount of the first enzyme is at least 1 mg for each mmol of polyethylene glycol of general formula (II); preferably the amount of the first enzyme ranges from 1 mg to 150 mg for each mmol of polyethylene glycol of general formula (II); more preferably the amount of the first enzyme ranges from 1 mg to 30 mg for each mmol of polyethylene glycol of general formula (II); even more preferably the amount of the first enzyme ranges from 2 mg to 15 mg for each mmol of polyethylene glycol of general formula (II); even more preferably the amount of the first enzyme ranges from 3 mg to 13 mg for each mmol of polyethylene glycol of general formula (II).

[0078] embodiment for better results, the amount of the second enzyme is at least 1 mg for each mmol of compound of general formula (VI) or compound of general formula (VI'); preferably the amount of the second enzyme ranges from 1 mg to 150 mg for each mmol of compound of general formula (VI) or compound of general formula (VI'); more preferably the amount of the second enzyme ranges from 1 mg to 30 mg for each mmol of compound of general formula (VI) or compound of general formula (VI'); even more preferably the amount of the second enzyme ranges from 2 mg to 15 mg for each mmol of compound of general formula (VI) or compound of general formula (VI'); even more preferably the amount of the second enzyme ranges from 3 mg to 13 mg for each mmol of compound of general formula (VI) or compound of general formula (VI'). [0079] In an embodiment for better results, the activity of the first enzyme, the activity of the second enzyme or the or the activity of the third enzyme ranges from 100 U/g to 20000 U/g; preferably 200 U/g to 15000 U/g; more preferably 250 U/g to 10000 U/g.

[0080] In an embodiment for better results, the enzymatic mono esterification is performed at a temperature ranging from 0°C - 120°C; preferably 40°C -70°C; more preferably 50°C -60°C; even more preferably 55°C.

[0081] In an embodiment for better results, the enzymatic mono esterification is performed in a pH ranging from 1 to 8; preferably 2-6.

[0082] In an embodiment for better results, the first suitable organic solvent is selected from the group consisting of: heptane, ketone, methyl isobutyl ketone, acetonitrile, tetrahydrofuran, toluene, tert-amyl alcohol, ethyl acetate, diethyl ether, dioxane, dimethylsulfoxide, cyclohexanone, methyl ethyl ketone, isopropyl alcohol, Methyl tertbutyl alcohol, diisopropylalcohol, methyltetrahydrofuran, anisole, or mixtures thereof; preferably methyl isobutyl ketone.

[0083] In an embodiment for better results, the second, third, fourth, fifth or sixth suitable organic solvent are independently selected from the group consisting of: dichloromethane, tetrahydrofuran, diethyl ether, Isopropylether, 2-methyl tetrahydrofuran, tetrahydrofuran, Dioxane, Methyl tert-Butyl Ether, Ethylene Glycol Dimethyl Ether, Ethyl Acetate, Methyl Acetate, Butyl Acetate, Isopropyl Acetate, Isopropyl Acetate, Acetone, Methyl Ethyl Ketone, Cyclohexanone, Methyl Isobutyl Ketone, Diisobutyl Ketone, isopropyl alcohol, Methyl tertbutyl alcohol, diisopropylalcohol, methyltetrahydrofuran, anisole; preferably selected from dichloromethane, tetrahydrofuran, diethyl ether, Isopropylether, 2-methyl tetrahydrofuran, tetrahydrofuran, or mixtures thereof.

[0084] In an embodiment for better results, the second suitable organic solvent, the third suitable organic solvent, the fourth suitable organic solvent, the fifth suitable organic solvent and the sixth suitable organic solvent is tetrahydrofuran.

[0085] In an embodiment for better results, the first base, the second base and the third base are independently selected from the list consisting of: sodium carbonate (NajCOs), Potassium carbonate (K2CO3), Sodium bicarbonate (NaHCOs), Potassium bicarbonate (KHCO3), Sodium methoxide (NaOCHs), Sodium ethoxide (NaOEt), Potassium tert-butoxide (KOtBu), Lithium diisopropylamide (LDA), Triethylamine (EtsN), Diisopropylethylamine (DIPEA), Pyridine, Sodium hydride (NaH), Potassium hydride (KH), l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), potassium bis(trimethylsilyl)amide; or mixtures thereof.

[0086] In an embodiment for better results, the first base, the second base and the third base is potassium bis(trimethylsilyl)amide.

[0087] In an embodiment for better results, the first suitable aqueous-based or first water miscible solvent and the second suitable aqueous-based or second water miscible solvent are independently selected from the group consisting of: water, ethanol, methanol, acetone, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, or mixtures thereof; preferably water.

[0088] In an embodiment for better results, the first suitable aqueous-based or first water miscible solvent and the second suitable aqueous-based or second water miscible solvent is water.

[0089] In an embodiment for better results, the method further comprises the step of reacting the compound of general formula (V) with a compound of general formula (VIII),

Formula (VIII) to obtain a compound of general formula (IX), in a fourth suitable organic solvent and in the presence of a second base, formula (IX) wherein: the reaction is performed at a temperature ranging from 24-120°C;

PG is a protecting group; preferably a protecting group selected from the group consisting of: benzyl ether, Tert-Butyldimethylsilyl, Trimethylsilyl, methyl ether tetrahydropyranyl, 4,4'- Dimethoxytrityl, p-Methoxybenzyl, benzyl; preferably benzyl.

[0090] In an embodiment, n" is at least 1.

[0091] In an embodiment for better results, n" ranges from 1-100; preferably 2-50; more preferably 4-

30.

[0092] In an embodiment for better results, the method further comprises the step of reacting the compound of general formula IX with a third enzyme, in a suitable second aqueousbased or second water miscible solvent, to obtain the compound of general formula (XV) formula (XV) wherein: the third enzyme is selected from the group consisting of: lipase, esterase, protease, transferase, ligase or mixtures thereof; the reaction is performed at a temperature ranging from 5-100°C. [0093] In an embodiment for better results, the amount of the third enzyme is at least 1 mg for each mmol of compound of general formula IX ; preferably the amount of enzyme ranges from 1 mg to 150 mg for each mmol of compound of general formula IX; more preferably the amount of enzyme ranges from 1 mg to 30 mg for each mmol of compound of general formula IX; even more preferably the amount of enzyme ranges from 2 mg to 15 mg for each mmol of compound of general formula IX ; even more preferably the amount of enzyme ranges from 3 mg to 13 mg for each mmol of compound of general formula IX .

[0094] In an embodiment for better results, the step of reacting the compound of general formula IX with a third enzyme, in a suitable second aqueous-based or second water miscible solvent, to obtain the compound of general formula (XV), is performed in a pH ranging from 1 to 8; preferably 2-6.

[0095] In an embodiment for better results, the first, the second or the third enzyme are independently selected from a lipase, esterase, protease, transferase or ligase obtained from a microorganism selected from the list consisting of: Alcaligenes spp, Aspergillus spp, Candida rugosa , Saccharomyces cerevisiae, Candida Antarctica, Chromobacterium spp, Rhizomucor spp, Penicilium spp, Pseudomonas spp, Rhizopus spp, Thermomyces spp, Geotrichum spp, Mucor spp, Burkholderia spp , Alcaligenes spp, Candida spp, Chromobacterium spp, , Bacillus subtilis, Rhizopus spp, Serratia marcescens, Escherichia coli, Pseudomonas fluorescens, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris, Schizosaccharomyces pombe, Pichia pastoris, Kluyveromyces lactis, Streptomyces spp, Bacillus cereus, Salmonella typhimurium, , Streptomyces spp, Staphylococcus spp, Pichia spp, Saccharomyces spp, Schizosaccharomyces spp, Schwanniomyces spp, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe or Fusarium solani pisi, or mixtures thereof.

[0096] In an embodiment for better results, the first, the second or the third enzyme are independently selected a lipase, esterase or protease obtained from a microorganism selected from the list consisting of: Candida spp, Thermomyses lanuginosus and Mucor miehei.

[0097] In an embodiment for better results, wherein the first, the second and/or the third enzyme is a lipase (triacylglycerol acyl hydrolases, EC number 3.1.1.3).

[0098] In an embodiment for better results, the first, the second and/ or the third enzyme is lipase enzyme obtained from Candida antarctica B; preferably Candida antarctica lipase B (CALB) immobilized on a hydrophobic carrier; more preferably Candida antarctica lipase B (CALB) immobilized on acrylic resin.

[0099] In an embodiment for better results, the first, the second and/or the third enzyme is lipase from Mucor miehei; preferably lipase from Mucor miehei immobilized on a hydrophobic carrier.

[00100] In an embodiment for better results, the method further comprises the step of: removing the protecting group (PG) from the compound of general formula (XV), in a fifth suitable organic solvent, to obtain a compound of general formula (I")

Formula (I").

[00101] In an embodiment for better results, the step of removing the protecting group (PG) is performed with palladium on carbon (Pd/C), preferably 5-10% Pd by weight (of the compound of formula (XV), under hydrogen atmosphere at temperature ranging from 25-50°C and pressure ranging from 1-30 bar.

[00102] In an embodiment for better results, the method further comprises the step of reacting the polyethylene glycol of general formula (I), polyethylene glycol of general formula (I') or polyethylene glycol of general formula (II'), with a leaving group (LG2), in a fifth suitable organic solvent, to obtain the compound of general formula (XII), compound of general formula (XII') or compound of general formula (XII"), respectively formula (XII) formula (XII') formula (XII") wherein:

LG2 is selected from p-toluenesulfonate or methanesulfonate; preferably p-toluenesulfonate; the reaction is performed at a temperature ranging from 25-100°C.

[00103] In an embodiment for better results, the method further comprises the step of: reacting a compound of general formula (XII), a compound of general formula (XII') or a compound of general formula (XII") with a compound of general formula (IV), in a sixth suitable organic solvent and in the presence of a third base, to obtain the compound of general formula (XIII), compound of general formula (XIII') or compound of general formula (XIII"), respectively, formula (XIII) formula (XIII') formula (XIII") wherein the reaction is performed at a temperature ranging from 24-120°C. [00104] Another aspect of the present disclosure relates to a method for obtaining a high molecular weight monodispersed polyethylene glycol of general formula (I'")

Formula (I'") comprising a step of enzymatic mono esterification of a polyethylene glycol derivative of general formula (X) with a sugar-based compound or a lipid-based compound formula (X) to obtain a mono esterified polyethylene glycol derivative of general formula (XI), formula (XI) wherein: the enzymatic mono esterification is performed in a seventh suitable organic solvent and catalysed by a fourth enzyme selected from the group consisting of: lipase, esterase, protease, transferase, ligase or mixtures thereof;

Rs is a sugar-based compound or a lipid-based compound; n is at least 1; preferably ranges from 1-100; p is 0 or at least 1.

[00105] In an embodiment for better results, the sugar-based compound is selected from the list consisting of: glucuronic acid, fructose, lactose, maltose, glucose, galactose, isosaccharinic acid, gluconic acid, glucaric acid, glycerol, xylitol, mannitol, ribose, deoxyribose, adenine, guanine or cytosine;

[00106] In an embodiment for better results, the lipid-based compound is cholesterol.

[00107] In an embodiment for better results, the seventh suitable organic solvent is selected from the group consisting of: heptane, ketone, methyl isobutyl ketone, acetonitrile, tetrahydrofuran, toluene, tert-amyl alcohol, ethyl acetate, diethyl ether, dioxane, dimethylsulfoxide, cyclohexanone, methyl ethyl

T1 ketone, isopropyl alcohol, methyl tertbutyl alcohol, diisopropylalcohol, methyltetrahydrofuran, anisole, or mixtures thereof; preferably methyl isobutyl ketone.

[00108] In an embodiment for better results, the method of the present disclosure is performed at a pressure at or above atmosphere pressure; preferably equal or above 101.32500 kPa (1 atmosphere).

[00109] In an embodiment for better results, the method of the present disclosure further comprises a step of purifying the polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I').

[00110] In an embodiment for better results, the method of purifying the polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I') comprises the following steps: providing an aqueous mixture comprising a suitable aqueous-based or water miscible solvent, the polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I') and one or more impurities; contacting the aqueous mixture with a deep eutectic solvent comprising a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA), wherein the deep eutectic solvent selectively dissolves said impurities; separating the aqueous phase comprising the purified polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I'); and optionally recovering the purified polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I') by precipitation, filtration, or evaporation.

[00111] In an embodiment for better results, the method of the present disclosure is performed in batch or in continuous process.

[00112] Another aspect of the present disclosure relates to a stable high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (II') obtainable by the method described in the present disclosure.

[00113]The PEG obtained by the method of present disclosure is non-toxic and non-immunogenic, stable and with an improved polydispersity index. In an embodiment, the PEG of the present disclosure has a molecular of at least 50 g/mol; preferably at least 150 g/mol; with a purity before purification of at least 90%; preferably 95%; and an improved polydispersity index.

[00114] Measurement of the polydispersity index (PDI) can be carried out in a number of ways, in the present disclosure Gel Permeation Chromatography (GPC), also known as Size Exclusion Chromatography (SEC), is used to assess the polydispersity index (PDI) of the monodisperse PEG. This technique separates PEG molecules based on their hydrodynamic volume, allowing us to determine the molecular weight distribution. The GPC system is calibrated using PEG standards of known molecular weights to ensure accurate measurement. The PDI is calculated as the ratio of the weight-average molecular weight (Mw) to the number-average molecular weight (Mw), providing a quantitative measure of the distribution of molecular weights in the sample.

[00115] Another method to measure PDI includes the use of mass Spectrometry (MS) that is used to confirm the molecular weight and identify any impurities.

[00116] High-performance liquid Chromatography (HPLC) and NMR (Nuclear Magnetic Resonance) were used to measure the purity of the monodisperse polymer.

[00117] In an embodiment for better results, the polyethylene glycol (stable high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') obtainable by the method described in the present disclosure is selected from the list consisting of: PEG195, PEG370, PEG679, PEG722, PEG2000, PEG6000.

[00118] In the context of the present disclosure, a polyethylene glycol (PEG)(number) of general formula (I) or general formula (I') refers to a PEG having a molecular weight, expressed in grams per mole (g/mol), equal to the indicated number. For example, PEG6000 designates a PEG of general formula (I) or general formula (I') with a molecular weight of 6000 g/mol.

[00119] In an embodiment for better results, the polydispersity index (PDI) of the obtained polyethylene glycol ranges from 1.0 - 1.1; preferably 1.00 - 1.04; more preferably 1.00 - 1.02; more preferably 1.00 - 1.01.

[00120] In an embodiment for better results, the polydispersity index (PDI) of the obtained polyethylene glycol ranges from 1.00 to 1.10; preferably 1.00 to 1.08.

[00121] In an embodiment for better results, In an embodiment for better results, the polydispersity index (PDI) of the obtained polyethylene glycol ranges from 1.000 - 1.005; preferably 1.000 - 1.002; more preferably 1.000 - 1.001.

[00122] Another aspect of the present disclosure relates to the use of the stable high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') herein described for use in medicine, preferably as a pharmaceutical acceptable excipient, including drug delivery systems, hydrogels for wound dressings, protein and/or peptide conjugation.

[00123] Another aspect of the present disclosure relates to the use of the stable high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') herein described for use in medicinal devices, preferably as lubricant for medical devices, or coatings for implants.

[00124] Another aspect of the present disclosure relates to the use of the stable high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') herein described as a cosmetic acceptable excipient for cosmetical formulations or devices. [00125] Another aspect of the present disclosure relates to the use of an enzyme selected from the group consisting of: lipase, esterase, protease, transferase, ligase or mixtures thereof, as a biocatalyst for mono esterification of a polyethylene glycol or a polyethylene glycol derivative.

[00126] Another aspect of the present disclosure relates to a deep eutectic solvent (DES) comprising a mixture of a carboxylic acid and a hydrogen bond acceptor in a molar ratio ranging from 1:0.2 to 1:5, wherein: the carboxylic acid is selected from: the hydrogen bond acceptor is selected from the group consisting of: menthol, terpineol, geraniol, linalol, methyltriphenylphosphonium, thymol, limonene, terpinene, eugenol, hinokitiol, carvone, pirene, coumarin, 6-methyl-coumarin, lidocaine, nicotinic acid, humulene, taxadiene, geosmin, ocimene, nerolidol, farnesol, tetra ammonium chloride, proline, alanine, choline, or mixtures thereof.

[00127] Unexpectedly, the isolation of PEG with increased chain length can be efficiently performed through in situ formation of a novel eutectic solvent by separating the related acid by-product into a second layer. This approach offers an efficient, sustainable, and environmentally friendly alternative to conventional separation and purification techniques. It was surprisingly found that the DES described in the present disclosure allow simple and efficient purification of the monodispersed PEG'S synthetized, in a reproducible manner and on an industrial scale.

[00128] The deep eutectic solvent of the present disclosure allows to obtain a PEG with a polydispersity index ranging from 1.00 to 1.10; preferably 1.00 to 1.08.

[00129] In a preferred embodiment, the deep eutectic solvent of the present disclosure allows to obtain a PEG with a polydispersity index ranging from 1.000 - 1.005; preferably 1.000 - 1.002; more preferably 1.000 - 1.001.

[00130] The deep eutectic solvent employed in the present disclosure allows to obtain a polyethylene glycol with a high degree of purity, exceeding 95%, preferably exceeding 97%, more preferably exceeding 98%, and most preferably exceeding 99%.

[00131] In an embodiment for better results, the deep eutectic solvent comprises: a mixture of a carboxylic acid and a hydrogen bond acceptor in a molar ratio ranging from 1:0.2 to 1:5 (carboxylic acid: hydrogen bond acceptor), wherein: the carboxylic acid is selected from the list consisting of: 3-aminophenylacetic acid, 4- aminophenylacetic acid, 2-aminophenylacetic acid, 3-nitrophenylacetic acid, 4-nitrophenylacetic acid, 2-nitrophenylacetic acid, 3- nitrobenzoic acid, 4- nitrobenzoic acid, 2-nitrobenzoic acid, 3- methoxyphenylacetic acid, 4-methoxyphenylacetic acid, 2-methoxyphenylacetic acid, 3- methylphenylacetic acid, 4-methylphenylacetic acid, 2-methylphenylacetic acid, 3- methoxycinnamic acid, 2-(tiophen-3-yl)acetic acid, (E)-5-methoxypent-4-enoic acid, 2-(furan-3- yl)acetic acid, 2-(quinolin-3-yl)acetic acid, 2-(3-methoxyphenyl)-2-oxoacetic acid, 3-butynoic acid, maleimidyl acetic acid, pyridin-3-yl acetic acid, pyridin-4-yl acetic acid, maleimidyl propionic acid, 3- acetamidophenylacetic acid, 3-methoxycinnamic acid, methyl-(3-methoxy)cinnamate, triphenyl acetic acid, diphenyl acetic acid, 3-hydroxy phenylacetic acid, 4-hydroxy phenylacetic acid, 2- hydroxy phenylacetic acid, phenyl acetic acid, glucuronic acid, isosaccharinic acid, gluconic acid or glucaric acid. the hydrogen bond acceptor is selected from the group consisting of: menthol, terpineol, geraniol, linalol, methyltriphenylphosphonium, thymol, limonene, terpinene, eugenol, hinokitiol, carvone, pirene, coumarin, 6-methyl-coumarin, lidocaine, nicotinic acid, humulene, taxadiene, geosmin, ocimene, nerolidol, farnesol, tetra ammonium chloride, proline, alanine, choline, or mixtures thereof.

[00132] In an embodiment for better results, the carboxylic acid is selected from the list consisting of: 3- aminophenylacetic acid, 4-aminophenylacetic acid, 2-aminophenylacetic acid, 3-nitrophenylacetic acid, 4-nitrophenylacetic acid, 2-nitrophenylacetic acid, 3- nitrobenzoic acid, 4- nitrobenzoic acid, 2- nitrobenzoic acid, 3-methoxyphenylacetic acid, 4-methoxyphenylacetic acid, 2-methoxyphenylacetic acid, 3-methylphenylacetic acid, 4-methylphenylacetic acid, 2-methylphenylacetic acid, 3- methoxycinnamic acid, 2-(tiophen-3-yl)acetic acid, (E)-5-methoxypent-4-enoic acid, 2-(furan-3-yl)acetic acid, 2-(quinolin-3-yl)acetic acid, 2-(3-methoxyphenyl)-2-oxoacetic acid, 3-butynoic acid, maleimidyl acetic acid, pyridin-3-yl acetic acid, pyridin-4-yl acetic acid, maleimidyl propionic acid, 3- acetamidophenylacetic acid, 3-methoxycinnamic acid, methyl-(3-methoxy)cinnamate, triphenyl acetic acid, diphenyl acetic acid, 3-hydroxy phenylacetic acid, 4-hydroxy phenylacetic acid, 2-hydroxy phenylacetic acid, phenyl acetic acid, glucuronic acid, isosaccharinic acid, gluconic acid or glucaric acid.

[00133] In an embodiment for better results, the carboxylic acid is selected from the list consisting of: 2- (m-tolyl)acetic acid, 3-methoxyphenylacetic acid, methyl-3-methoxy cinnamate, glucuronic acid or maleimidyl acetic acid.

[00134] In an embodiment for better results, the carboxylic acid is 3-methoxyphenyl acetic acid.

[00135] In an embodiment for better results, the hydrogen bond acceptor is selected from the list consisting of: coumarin, menthol, thymol, choline chloride, 6-methyl coumarin or tetrabutylammonium chloride.

[00136] In an embodiment for better results, the hydrogen bond acceptor is thymol.

[00137] In an embodiment for better results, the molar ratio of the carboxylic acid and the hydrogen bond acceptor ranges from 1:0.2 to 1:5; preferably 1:0.5 to 1:2; more preferably 1:1.

[00138] In an embodiment for better results, the deep eutectic solvent comprises a mixture of 3- methoxy phenylacetic acid and thymol in a molar ratio ranging from 1:0.2 to 1:5; preferably, comprises a mixture of 3-methoxy phenylacetic acid and thymol in a molar ratio ranging from 1:0.5 to 1:2; more preferably, comprises a mixture of 3-methoxy phenylacetic acid and thymol in a molar ratio of 1:1.

[00139] In an embodiment for better results, the deep eutectic solvent comprises a mixture of 3- methoxy phenylacetic acid and thymol in a molar ratio of 1:1.

[00140] In an embodiment for better results, the deep eutectic solvent comprises: a mixture of 3- methoxy phenylacetic acid and coumarin in a molar ratio of 1:1; or a mixture of 3-methoxy phenyl acetic acid and menthol in a molar ratio of 1:3; or a mixture of 3-methoxy phenyl acetic and thymol in a molar ratio of 1:1; or a mixture of 3-methoxy phenyl acetic and choline chloride in a molar ratio of 3:1; or a mixture of 3-methoxy phenyl acetic and 6-methyl coumarin in a molar ratio of 1:1; or a mixture of 3- methoxy phenyl acetic and tetrabutylammonium chloride in a molar ratio of 1:1.

[00141] In an embodiment for better results, the deep eutectic solvent comprises: a mixture of 3- methoxy phenylacetic acid and lidocaine in a molar ratio of 1:1; or a mixture of 3-methoxy phenyl acetic acid and methyl- triphenylphosphonium bromide in a molar ratio of 1:2.2; or a mixture of 3-methyl phenyl acetic acid and lidocaine in a molar ratio of 1:1.5; or a mixture of 3-methyl phenyl acetic acid and menthol in a molar ratio of 1:1.2; or a mixture of 3-methyl phenyl acetic acid and thymol in a molar ratio of 1:1; or a mixture of 3-methyl phenyl acetic acid and coumarin in a molar ratio of 1:2; or a mixture of 3-aminophenyl acetic acid and choline chloride in a molar ratio of 1:1.3; or a mixture of 3-aminophenyl acetic acid and thymol in a molar ratio of 1:1; or a mixture of 3-aminophenyl acetic acid and 6- methylcoumarin in a molar ratio of 1:1.4; or a mixture of 3-aminophenyl acetic acid and coumarin in a molar ratio of 1:1.4; or a mixture of 3-aminophenyl acetic acid and lidocaine in a molar ratio of 1:1.5; or a mixture of phenyl acetic acid and Thymol in a molar ratio of 1:1.2; or a mixture of phenyl acetic acid and Menthol in a molar ratio of 1:1.2; or a mixture of phenyl acetic acid and Coumarin in a molar ratio of 1:1.2; or a mixture of phenyl acetic acid and 6-methylcoumarin in a molar ratio of 1:1.2; or a mixture of phenyl acetic acid and Lidocaine in a molar ratio of 1:1.

[00142] Another aspect of the present disclosure relates to the use of the deep eutectic solvent herein described as a purifier of mono dispersive polyethylene glycol; preferably a high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I').

[00143] Another aspect of the present disclosure relates to the use of the deep eutectic solvent herein described as a carboxylic acid extractor, preferably a carboxylic acid of general formula III.

[00144] Another aspect of the present disclosure relates to the use of the deep eutectic solvent herein described for extracting a carboxylic acid selected from the group consisting of: 3-aminophenylacetic acid, 4-aminophenylacetic acid, 2-aminophenylacetic acid, 3-nitrophenylacetic acid, 4-nitrophenylacetic acid, 2-nitrophenylacetic acid, 3- nitrobenzoic acid, 4- nitrobenzoic acid, 2-nitrobenzoic acid, 3- methoxyphenylacetic acid, 4-methoxyphenylacetic acid, 2-methoxyphenylacetic acid, 3- methylphenylacetic acid, 4-methylphenylacetic acid, 2-methylphenylacetic acid, 3-methoxycinnamic acid, 2-(tiophen-3-yl)acetic acid, (E)-5-methoxypent-4-enoic acid, 2-(furan-3-yl)acetic acid, 2-(quinolin-3- yl)acetic acid, 2-(3-methoxyphenyl)-2-oxoacetic acid, 3-butynoic acid, maleimidyl acetic acid, pyridin-3-yl acetic acid, pyridin-4-yl acetic acid, maleimidyl propanoic acid, 3-acetamidophenylacetic acid, 3- methoxycinnamic acid, methyl-(3-methoxy)cinnamate, triphenyl acetic acid, diphenyl acetic acid, 3- hydroxy phenylacetic acid, 4-hydroxy phenylacetic acid, 2-hydroxy phenylacetic acid, phenyl acetic acid, glucuronic acid, isosaccharinic acid, gluconic acid or glucaric acid; preferably 2-(m-tolyl)acetic acid, 3- methoxyphenylacetic acid, methyl-3-methoxy cinnamate, glucuronic acid and maleimidyl acetic acid; more preferably 3-methoxyphenyl acetic acid.

[00145] In an embodiment for better results, the method for obtaining an extended high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') further comprises the step of: adding the deep eutectic solvent herein described to the aqueous mixture comprising the suitable aqueous-based or water miscible solvent, the polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I'), and one or more impurities; obtain a first liquid phase comprising the aqueous solvent and the polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I') and a second liquid phase comprising the deep eutectic solvent and one or more impurities; separating and collecting the aqueous phase comprising the polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I').

[00146] According to one aspect of the present disclosure, there are processes provided for preparing polyethylene glycol comprising: a) monosubstitution of PEG: reacting polyethylene glycol with a carboxylic acid or its derivative in the presence of an enzyme in a solvent to form polyethylene glycol monoester, Ester-PEG (IV); b) addition of leaving group: preparing Ester-PEG-LG (V) from Ester-PEG (IV) or preparing LG-PEG- LG (XII) from PEG; c) chain extension: reacting Ester-PEG-LG (V) with Ester-PEG (IV) in the presence of a base to form Ester-PEGzn-Ester (VI); or reacting Ester-PEG-LG (V) with PG-PEG (VIII) in the presence of a base to form Ester-PEG2n-PG (IX); or reacting Ester-PEG (IV) with LG-PEG-LG (XII) in the presence of a base to form Ester-PEG2n+_P-Ester (XIII). d) deprotection of Ester-PEG2n-Ester (VI) or Ester-PEG2n+_P-Ester (XIII) by applying an enzyme in the presence of water in organic solvents, aqueous solvents, aqueous based solvents, or mixtures thereof resulting in PEG2n or PEG2n+_P (I) and the related acid; or the removal of the Ester group of Ester-PEG2n-PG (IX) by applying an enzyme resulting in PG-PEG (XV) and the removal of PG group of PG-PEG. The isolation of PEG with increased chain length is performed through a novel eutectic solvent preferentially formed in situ by separating the related acid by-product into a second layer.

[00147] According to another aspect of the present disclosure, there is provided a process comprising: a) monosubstitution of PEG: reacting polyethylene glycol with a carboxylic acid or its derivative in the presence of an enzyme in a solvent to form polyethylene glycol monoester, Ester-PEG (IV).

[00148] According to a further aspect of the present disclosure, there is provided a process comprising: d) in the deprotection step the isolation of PEG with increased chain length is performed through in situ formation of a novel eutectic solvent by separating the related acid by-product into a second layer.

[00149] Other aspects of the present disclosure relate to polyethylene glycol obtainable by the processes of the present disclosure. [00150] Other aspects of the present disclosure relate to polyethylene glycol heteroderivatives obtainable by the processes of the present disclosure.

[00151] Surprisingly, it has been found that step a) of the present disclosure affords the key intermediate polyethylene glycol monoester (Ester-PEG) in higher yields (equal or higher than 65%) and higher selectivity (typically equal or higher than 95%) than the process disclosed by Ahmed et al. ³⁴ without needing to perform purification by column chromatography as in EP1594440B1. Step a) of the present disclosure unforeseen controls the formation of undesirable disubstitution impurity, polyethylene glycol diester (Ester-PEG-Ester).

[00152] Additionally, it has been unexpectedly found that the isolation of PEG with increased chain length is performed with high efficiency through in situ formation of a novel eutectic solvent by separating the related acid by-product into a second layer.

[00153] The formation of deep eutectic solvents (DES) in situ represents a novel approach, wherein the hydrogen bond donor (HBD) and hydrogen bond acceptor (HBA) are introduced into an aqueous solution, thereby facilitating the formation of DES in situ, thereby enhancing extraction efficiency optimization. In the present disclosure, the sub product (that can act as HBD), formed during rection, is used in combination with a HBA to form a DES in situ.

[00154] Therefore, our finding offers a more efficient and sustainable way to separate PEG from its protecting group by using a novel eutectic solvent further contributing towards our approach innovative and unique features. This not only enhances the compound's synthesis but also fosters economic and environmental benefits through a reduced dependence on traditional solvents, thereby promoting the industrialization of sustainable practices. In summary, this process effectively addresses challenges linked to current reported methods, positioning it as a significant advancement in monodisperse PEG synthesis with promising prospects for industrial scalability.

DETAILED DESCRIPTION

[00155] The present disclosure relates to novel processes for the preparation of polyethylene glycols (PEG) and their derivatives. Additionally, the disclosure describes the characteristics of the obtained PEG products.

[00156] As used herein, the term "high molecular weight polyethylene glycol" refers to a polyethylene glycol having a molecular weight of at least 50 g/mol; preferably ranging from 150 g/mol - 20 000 g/mol; more preferably 195 g/mol - 10000 g/mol.

[00157] As used herein, the term "stable" in "stable high molecular weight monodispersed polyethylene glycol" refers to a polyethylene that retains its chemical integrity, molecular weight distribution, polydispersity index and functional performance characteristics over at least 12 months under 25°C and 60% RH (relative humidity); preferably at least 24 months; more preferably at least 36 months. Preferably a "stable high molecular weight monodispersed polyethylene glycol" exhibits less than 5 % variation in the polydispersity index value, in comparison with the polydispersity index value in t=0 months (after manufacturing).

[00158] As used herein, the term monodispersed in "monodispersed polyethylene glycol" refers to a polyethylene glycol polymer sample in which the constituent polymer chains exhibit a narrow molecular weight distribution, such that the polydispersity index (PDI) is less than or equal to 1.10, preferably less than or equal to 1.08, and most preferably approaching 1.00.

[00159] Polydispersity index (PDI) is used as a measure of broadness of molecular weight distribution. The larger the PDI, the broader the molecular weight. PDI of a polymer is calculated as the ratio of weight-average molecular weight (Mw) by number average molecular weight (Mn):

Polydispersity index=Mw/Mn.

[00160] The term "extended", as used herein in the expression "extended high molecular weight monodispersed polyethylene glycol", refers to a polyethylene glycol (PEG) product wherein the polymer chain has been lengthened relative to the PEG(s) used as starting material(s). Specifically, the term denotes that the resulting PEG possesses a higher degree of polymerization and increased molecular weight compared to the initial PEG reactants, as a result of a controlled chain-extension process.

[00161]This extension maintains a predominantly linear architecture and narrow molecular weight distribution (low polydispersity index).

[00162] The first step of the present disclosure describes a novel method for transforming polyethylene glycol (PEG) into its monosubstituted derivative using an enzyme. The reaction between one of the terminal hydroxyl groups in PEG and a carboxylic acid or its derivative in the presence of an enzyme yields polyethylene glycol monoester, Ester PEG (I) with at least 90% purity, preferentially equal or higher than 95% purity relative to the disubstitution impurity.

Scheme 4: PEG monosubstitution

[00163] The second step of the present disclosure involves the addition of one or two leaving groups by applying one of the approaches described below:

The addition of a leaving group to Ester-PEG (IV) forming Ester-PEG-LGi (V) via a chemical transformation suitable for the LGi described in the literature.

Scheme 5: Addition of leaving group - approach 1.

The addition of leaving groups to both terminal hydroxyl groups in PEG forming LG2-PEG-LG2 (XII) via a chemical transformation suitable for the LG2 described in the literature.

Solvent

LG-PEG-LG (XII)

Scheme 6: Addition of leaving group - approach 2.

[00164] "Leaving group" (LG) (LGi or LG2), as used herein, means a chemical moiety that can be or is displaced by a nucleophile to form a new chemical bond, generally via an S_N2-type displacement mechanism.

[00165]The preferred leaving group of the present disclosure is the p-toluenesulfonate or Ts group. However, others known in the art, like the Ms (methanesulfonate), also are useful.

[00166] The third step of the present disclosure involves the extension of the chain length from n to 2n or 3n by applying one of the approaches described below:

• The formation of elongated polyethylene glycol diester, Ester-PEG2n-Ester (VI) by reacting Ester-PEG-LG (V) with Ester-PEG (IV) in the presence of a base.

Ester-PEG-LG (V) Ester-PEG (IV) Ester-PEG-Ester (VI)

Scheme 7: Chain elongation reaction - approach 1

• The formation of elongated polyethylene glycol monoester monoalkoxyl or polyethylene glycol monoester monoaryloxyl, Ester-PEG_n+n--PG (IX) by reacting Ester-PEG-LGi (V) with PG- PEG (VIII) in the presence of a base, where PG corresponds to protecting group. Protecting group (PG), as used herein, means a molecular group that blocks a functional group from reacting during other chemical operations/transformations. A PG is inert to these chemical operations/transformations. After the chemical transformations are complete, the PG can be removed or cleaved by specific chemical means such that it liberates the original functional group for further reaction. The chemical selectivity and the physical design of protecting groups are important to the present disclosure. There are a wide variety of protecting groups available and known in the art. Many of them can be used in the present disclosure. Base o Solvent O

Ester-PEG-LG (V) PG-PEG (VIII) Ester-PEG-PG (IX)

Scheme 8: Chain elongation reaction - approach 2.

• The formation of elongated polyethylene glycol diester, Ester-PEGan-Ester (XIII) by reacting LG2-PEG-LG2 (XII) with two molecules of Ester-PEG (IV) in the presence of a base.

LG-PEG-LG (XII) Ester-PEG (IV) Ester-PEG-Ester (XIII)

Scheme 9: Chain elongation reaction - approach 3.

[00167] The last step of the present disclosure involves the removal of the terminal functional groups to obtain the polyethylene glycol with extended chain length by applying one of the approaches depending on the terminal functional groups described below:

• The deprotection of Ester-PEG2n-Ester (VI) or Ester-PEGan-Ester (XIII) by applying an enzyme in the presence of water resulting in PEG2n or PEGan and the related acid.

Ester-PEG-Ester (VI) PEG

Scheme 10: Deprotection - approach 1

Ester-PEG-Ester (XIII) PEG

Scheme 11: Deprotection - approach 1

• The removal of the Ester group of Ester-PEG-PG (IX) by applying an enzyme resulting in PG- PEG (XV). PG-PEG enables further elongation without requiring the first step. The removal of PG group of PG-PEG via a chemical transformation suitable for PG described in the literature. Enzyme HO J- PG Reagent HO

■o OT„_tn, o H₂O Solvent PG (IX) PG-P PEG

Ester-PEG- EG (XV)

Scheme 12: Deprotection - approach 2

[00168]The isolation of PEG with increased chain length is performed through in situ formation of a novel eutectic solvent by separating the related acid by-product into a second layer. This approach offers an efficient, sustainable, and environmentally friendly alternative to conventional separation and purification techniques.

[00169] The present disclosure pertains to a novel method for the preparation of monodisperse polyethylene glycols (PEGs) and their derivatives, employing both batch and continuous processes. The method for synthesizing monodisperse PEG comprises the following sequential steps: a. Monosubstitution of PEG; b. Addition of leaving group; c. Chain extension; d. Deprotection; e. Isolation.

[00170] Step a) may be carried out as follows:

Reacting an alkyl or aryl carboxylic acid with a PEG monomer in a solvent, at a temperature between 0°C to about 120°C, with an enzyme to form ester-PEG. A salt can be used in the reaction. The solvent used in step a) may be selected from the following list of solvents: ketone, methyl isobutyl ketone, acetonitrile, tetrahydrofuran, toluene, tert-amyl alcohol, ethyl acetate, diethyl ether, dioxane, dimethylsulfoxide, cyclohexanone, methyl ethyl ketone or a combination of these, preferably methyl isobutyl ketone.

[00171] The substrate used in a) may be selected from the aryl or alkyl-based carboxylic acid-containing molecules, encompassing various substitutions such as alkyl, amino, nitro, methoxy, methyl, hydroxyl, and/or halogens (Table 1) and the PEG monomer (Scheme 13) that can be from MW 195 - 10 000 with alcohol terminal thus forming an ester bound between both substrates, resulting in Ester-PEG195 with 95% purity. Ester-PEG can also be made with a combination of ester and PEG monomer. The ester can be either from the list in Table 1, preferably (3-methoxy) phenylacetate, and a PEG monomer that can be from MW 195 - 10000, thus forming Ester-PEG with 75% conversion and 93% purity.

[00172] From Table 1 above, the list of derivatives can include benzoic acid, amino-substituted phenylacetic acids, including 3-, 4-, and 2-aminophenylacetic acids, nitro-substituted phenylacetic acids, including 3-, 4-, and 2-nitrophenylacetic acids, nitro-substituted benzoic acids, including 3-, 4-, and 2- nitrobenzoic acids, methoxy-substituted phenylacetic acids, including 3-, 4-, and 2-methoxyphenylacetic acids, methyl-substituted phenylacetic acids, including 3-, 4-, and 2-methylphenylacetic acids, 3- methoxycinnamic acid, 2-(tiophen-3-yl)acetic acid, (E)-5-methoxypent-4-enoic acid, 2-(furan-3-yl)acetic acid, 2-(quinolin-3-yl)acetic acid, 2-(3-methoxyphenyl)-2-oxoacetic acid, 3-butynoic acid, maleimidyl acetic acid, pyridin-3-yl acetic acid, pyridin-4-yl acetic acid, maleimidyl propionic acid, maleic anhydride, 3-acetamidophenylacetic acid, 3-methoxycinnamic acid, methyl-(3-methoxy)cinnamate, triphenyl acetic acid, diphenyl acetic acid, hydroxyl-substituted phenylacetic acids, including 3-, 4-, and 2-hydroxy phenylacetic acids, and phenyl acetic acid. Preferably the carboxylic acid chosen is 3-methoxyphenyl acetic acid. n = 0-136

R, = OH, COOH, NH₂

Scheme 13: PEG monomer.

[00173] In other embodiments, when PEG monomer end groups are COOH (Scheme 13), a combination with a variety of substrates from Table 2 is possible and with substrate a) from Table 1. If the PEG monomer end group is OH, a reaction with substrates a), g), h), i) from Table 2 is possible to form the esterified monosubstituted product. The same is applied to the esterified substrates from Table 1, substrates b) c) and d). If the PEG monomer end group is NH2 when combined with a carboxylic acidcontaining substrate from Table 1 or Table 2 forms an amino-ester-PEG.

[00174]Table2: Substrates used for esterification - sugar-based.

[00175]Table 3: Substrates used for esterification - lipid-based.

[00176]Typical examples of suitable enzymes, which are not intended to limit the invention in any way, are hydrolases namely lipases and/or esterases and/or proteases of microorganisms selected from the group consisting of Alcaligenes, Aspergillus, Candida rugosa, Saccharomyces cerevisiae, Candida Antarctica, Chromobacterium, Rhizomucor, Penicilium, Pseudomonas, Rhizopus, Thermomyces, Geotrichum, Mucor, Burkholderia and mixtures thereof. Lipases and esterases and hydrolases from the organisms Alcaligenes, Candida, Chromobacterium, Penicilium, Pseudomonas, Bacillus subtilis, Rhizopus, Rhizomucor, Thermomyces, Serratia marcescens and Cutinase of Fusarium solani pisi. Are preferred because they are particularly active, Candida, Thermomyses lanuginosus and Mucor miehei and especially Candida antarctica B, being particularly preferred to give high selectivity of 95%yield and 95% Ester-PEG. Enzymes can also comprise transferases or ligases from microorganisms: Escherichia coli, saccharomyces cerevisiae, Pseudomonas fluorescens, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris, Bacillus subtilis, Schizosaccharomyces pombe, Pichia pastoris and Kluyveromyces lactis, Streptomyces, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium, and various species of Pseudomonas, Streptomyces and Staphylococcus, fungi Cells (eg Aspergillus), yeast (eg any species of Pichia, Saccharomyces, Schizosaccharomyces and Schwanniomyces (including Pichia pastoris, Saccharomyces cerevisiae or Schizosaccharomyces pombe)).

[00177] In an embodiment, Candida antarctica lipase B is commercially available, in the present application Candida antarctica lipase B was obtained from Novozymes.

[00178] In an embodiment, lipase from Mucor miehei is commercially available, in the present application lipase from Mucor miehei was obtained from Creative Enzymes.

[00179] In an embodiment, the enzyme is Lipase (IUBMB nomenclature: Triacylglycerol lipase; Systematic name: Triacylglycerol acyl hydrolase; Synonyms: Lipase, glycerol ester hydrolase, triacylglycerol ester hydrolase).

[00180] In an embodiment, step a) must be carried out at a temperature between 0°C and 120°C and about 55°C, preferably between 50 and 60°C.

[00181] In an embodiment, step a) can or not contain salts from the following group: potassium carbonate, potassium bicarbonate, sodium chloride, sodium carbonate, sodium bicarbonate, sodium sulfate, calcium carbonate, ammonium chloride, ammonium sulfate, sodium thiosulfate, potassium sulfate, calcium chloride, preferably potassium carbonate.

[00182] One of the advantages of using an immobilized enzyme for this purpose is that this type of reaction is more sustainable and straightforward avoiding the high formation of impurity bis-substituted PEG. This process was developed in batch and continuous flow where in both cases high conversions were achieved and isolation was possible (95% conversion in batch and in continuous flow). Moreover, the use of an immobilized enzyme eases a more straightforward separation step of the reaction mixture. Furthermore, by avoiding the use of harsh reagents we are making this process more environmentally friendly.

[00183] The residence time of reagents along a selected distance of the continuous flow reactor, which is associated with the monosubstitution, can vary from 1 minute to 30 minutes. The yield of monosubstitution may be about (range) 5% or more, preferably 50% or more, and more preferably 90% or more, and the chromatographic purity of the resultant reaction crude may be about 50% or more and more preferably 90% or more.

[00184] In some embodiments, the use of a continuous flow reactor provides the ability to maintain temperatures and pressures that are not readily attainable in batch processes. In some cases, the chemical reaction is performed at a temperature of at least 30 °C and, in some cases, greater. In some cases, the chemical reaction is performed at a pressure of at least 100 psi or, in some cases, greater. The flow-through system may be designed and fabricated to be capable of withstanding a wide range of solvents and chemical conditions, including high temperature, high pressure, exposure to various solvents and reagents, and the like. A variety of materials can be used to construct a plug flow continuous flow reactor, including glass, different types of polymers (such as PFA, ETFE, and PEEK), Hastelloy®, silicon carbide, stainless steel, and/or high-performance alloys. These reactors may be equipped with static mixing apparatus and can handle slurries that can withstand certain temperatures and/or pressures. When multiple reactors are used, they may be connected to allow for fluid communication. It's important to note that the reactors don't have to be directly attached to each other, but they should be in fluid communication. Additionally, the reaction profile may be largely unaffected by the volume of the fluid sample, which allows for scalability without significant changes to the reaction profile. The rate at which reagents flow through a continuous flow reactor can be adjusted, controlled, or altered depending on the specific chemical synthesis reaction being carried out. The flow rate can vary at different distances within the reactor, and the rate of one reaction step can impact the rate of subsequent steps. Pumps are used to control and adjust the flow rates.

[00185] In other embodiments, the use of a batch reactor involves a closed vessel where enzymes and substrates are mixed in predefined quantities. The reaction progresses under controlled conditions, allowing for efficient utilization of enzymes and precise control over reaction kinetics. Specialized equipment or procedural steps may enhance the efficiency of the batch process. In the batch reaction, the enzymes can be deployed in various configurations, including being released directly into the reactor, immobilized within the reactor, or incorporated into the reactor mix. Specifically, this implies that the enzymes have the flexibility to either operate freely in the reactor, be securely affixed within the reactor structure, or be integrated into the stirring mechanism of the reactor, providing diverse options for enzyme utilization and storage during the reaction process.

[00186] The Ester-PEG can be used to further elongate PEG chain through already known use of leaving groups, thus increasing PEG length in a monodisperse way. It can also be used to synthesize derivatives without the need for chromatographic separations. The free hydroxyl from monosubstituted PEG can react to form the activated derivative for PEGylation.

[00187]To elongate the PEG chain, through step b) it is necessary to add a leaving group to the previously obtained monosubstituted PEG. Step b) may be carried out as follows:

[00188] Reacting compound IV in a solvent, at a temperature between 25°C to about 120°C, with a leaving group. The leaving group is chosen from those known in the art. In the present disclosure it can be mesyl or mesylate (Ms) or tosyl or tosylate (Ts), where tosyl group is the preferred leaving group. While others may be as effective, the tosylate is a derivative that is very easily made from the alcohol, produce intermediates that are easy to process and purify, and are made from starting materials that are inexpensive and very pure, namely tosyl chloride or p-toluene sulfonyl chloride and triethylamine. Tosyl chloride also is an easy to handle solid. The preferred solvent for making the tosylates is methylene chloride, but others like ethyl acetate may be preferred when these reactions are performed at a process scale of manufacturing.

[00189] Step c) includes the i) chain extension reaction by combining compound IV with compound V forming compound VI and the bis ester elongated PEG. This step also comprises other approaches as ii) the combination of compound V and compound VIII into compound IX and approach iii) with the combination of compound IV and compound XII into XIII. All these approaches are made in a solvent, at a temperature between 24°C and 120°C with a base as described in prior art. The solvent used in step c) may be selected from the group consisting of cyclic ethers such as tetrahydrofuran (THF). Step d) of the present disclosure may be carried out as follows: d) reacting compound VI or compound XIII or compound IX in a solvent with an enzyme. The solvents used in the process may be common organic solvents, aqueous solvents, aqueous based solvents, water, or mixtures thereof. Any compatible solvent or solvent system can be used. The solvent systems may comprise mixtures of water-miscible organic solvents and water. They may also comprise water immiscible organic solvents in contact with water. Any specific combinations of the above listed solvents may be used. All of the reactions may not be optimally carried out in the same solvent or solvent system. The enzymes used are the same as step a) and the reaction shall be carried out at a temperature between 0°C and 100°C and about 40°C, preferably between 25 and 40°C. At temperatures below than 25°C promotes monodeprotection.

[00190] When product I is obtained as free PEG, it is required to perform its isolation from the removed acid in step e). Therefore, in a further embodiment of the present disclosure, the separation of the hydrolysed acid from the PEG is made with a novel eutectic solvent that extracts the acid, leaving all PEG in the aqueous phase. In a particular embodiment, the present disclosure provides a eutectic solvent for extracting the hydrolysed acid, wherein the solvent is a clear, stable, and fluid mixture consisting essentially of:

(a) acid used in enzymatic process as hydrogen bond donor;

(b) at least one hydrogen bond acceptor compound selected from the group consisting of polyols and organic acids; and

(c) water.

[00191] The hydrogen bond donor compound may be selected from the previous list of carboxylic acid used in step a), preferably 3-methoxy phenylacetic acid. The term "hydrogen bond donor" refers herein to a chemical structure containing a suitable hydrogen bond donor atom bearing one or more protons that are attached to a relatively electronegative atom, such as an oxygen atom or a nitrogen atom.

[00192] In an embodiment of the present disclosure, the critical molar ratio of HDB to the at least one hydrogen bond acceptor compound is from 1:5 and 5:1 and is preferably 1:1 in the case of 3-methoxy phenylacetic acid. In a further embodiment of the present disclosure, the proportion by weight of water in the DES are between 0.025% (w/w) and 0.25%(w/w) and after the extraction in water, the water content is from 1 to 50%(w/w), and preferably 15 to 30%(w/w).

In a further aspect, the present disclosure provides the use of a eutectic extraction solvent as previous defined for extracting acids from alcohols.

[00193] In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the hydrogen bond acceptor compound (HBA) is selected from the group from Table 4 as coumarin, menthol, thymol, tetrabutylammonium chloride, tetrametylammonium chloride, betaine, choline chloride, L-proline, lidocaine, methyltriphenylphosphonium bromide preferably thymol in proportion 1:1.

[00194]Table 4: Hydrogen bond acceptor compound (HBA)

[00195] The eutectic solvent of the present disclosure can be prepared in separate and added to the aqueous mixture of PEG and the HDB to extract the HDB and isolate PEG or it can be applied in situ. The process of in-situ refers to the addition of a molar proportion of hydrogen bond acceptor (HBA) to an aqueous mixture containing hydrogen bond donor (HBD). This results in the formation of a droplet of deep eutectic solvent (DES) in situ, which can selectively remove the HBD from the mixture, and subsequently isolate polyethylene glycol (PEG).

EXAMPLES

[00196]The present disclosure is now illustrated without limiting it by the following examples. Example 1: Synthesis of Ester-PEG195

[00197] In this embodiment, the reactor was charged with acetonitrile (1 mL), 2-(m-tolyl)acetic acid (0.16 mmol) and tetraethylene glycol (PEG4, 1.12 mmol). The temperature was set to 50 °C, and the Lipase B from Candida Antarctica (5000 u/g, 4 mg) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The aqueous phase was extracted 2 times with ethyl acetate (50 mL). The combined organic phase was concentrated to dryness under vacuum (52 mg, 99% yield, 91% Ester-PEG195).

Example 2.1: Synthesis of Ester-PEG195

[00198] In this embodiment, the reactor was charged with MIBK (1 L), 3-methoxyphenylacetic acid (0.14 mol) and tetraethylene glycol (PEG4, 1.01 mol). The temperature was set to 55 °C, and Lipase B from Candida Antarctica (5000 u/g, 11 g) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and was extracted 3 times with a 3%(w/w) NaCI aqueous solution previously adjusted to pH 1. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum. (37 g, 77%, 100% Ester-PEG195).

Example 2.2: Synthesis of Ester-PEG195

[00199] In this embodiment, the reactor was charged with MIBK (1.5 L), 3-methoxyphenylacetic acid (0.31 mol) and tetraethylene glycol (PEG4, 2.2 mol). The temperature was set to 57 °C, and Lipase A from Candida Antarctica (10000 u/g, 7 g) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and was extracted 3 times with a 3%(w/w) NaCI aqueous solution previously adjusted to pH 1. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum. (50.5 g, 56.5% yield, 100% Ester-PEG195).

Example 2.3: Synthesis of Ester-PEG195

[00200] In this embodiment, the reactor was charged with MIBK (2 mL), methyl-3-methoxy cinnamate (0.3 mmol) and tetraethylene glycol (PEG4, 0.3 mmol). The temperature was set to 55 °C, and the Lipase from Mucor Mieihei (250 u/g, 40 mg) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and was extracted 3 times with a 3%(w/w) NaCI aqueous solution previously adjusted to pH 1. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum. (HPLC analysis: 36% consumption of starting raw material into 82% Ester-PEG195).

Example 2.4: Synthesis of Ester-PEG195

[00201] In this embodiment, the reactor was charged with MIBK (1 L), 3-methoxyphenylacetic acid (0.14 mol) and tetraethylene glycol (PEG4, 1.01 mol). The temperature was set to 55 °C, and Lipase from from Thermomyces Lanuginosus ((250 u/g, 11 g) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and was extracted 3 times with a 3%(w/w) NaCI aqueous solution previously adjusted to pH 1. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum. (HPLC analysis: 10% consumption of starting raw material into 100% Ester-PEG195).

Example 2.5: Synthesis of Ester-PEG195

[00202] In this embodiment, the reactor was charged with MIBK (1 L), 3-methoxyphenylacetic acid (0.14 mol) and tetraethylene glycol (PEG4, 1.01 mol). The temperature was set to 55 °C, and Transferase from E. coli (11 g) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and was extracted 3 times with a 3% (w/w) NaCI aqueous solution previously adjusted to pH 1. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum.

Example 2.6: Synthesis of Ester-PEG195

[00203] In this embodiment, the reactor was charged with MIBK (1 L), 3-methoxyphenylacetic acid (0.14 mol) and tetraethylene glycol (PEG4, 1.01 mol). The temperature was set to 55 °C, and Ligase from E. coli (11 g) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and was extracted 3 times with a 3%(w/w) NaCI aqueous solution previously adjusted to pH 1. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum.

Example 2.7: Synthesis of Ester-PEG195

[00204] In this embodiment, the reactor was charged with MIBK (1.5 L), 3-methoxyphenylacetic acid (0.31 mol) and tetraethylene glycol (PEG4, 2.2 mol). The pH was set to 4.45 with a solution of 1.2 mM of potassium bicarbonate. The temperature was set to 57 °C, and Lipase B from Candida Antarctica (5000 u/g, 7 g) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC and the final pH was 5.54. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and was extracted 3 times with a 3% (w/w) NaCI aqueous solution previously adjusted to pH 1. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum. .

(HPLC analysis: 85% consumption of starting raw material into 91% Ester-PEG195).

Example 3: Synthesis of Ester-PEG370

[00205] In this embodiment, the reactor was charged with MIBK (10 mL), 3-methoxyphenylacetic acid (6 mmol) and octaethylene glycol (PEG370, 42 mmol). The temperature was set to 50 °C, and the enzyme (40 mg) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The aqueous phase was extracted 2 times with ethyl acetate (50 mL). The combined organic phase was concentrated to dryness under vacuum (2.3 g, 88% yield, 94% Ester-PEG370).

Example 4: Synthesis of Ester-PEG195 in continuous flow

[00206] A solution of 3-methoxyphenylacetic acid (1 g, 5.5 mmol) and tetraethyleneglycol (6.67 mL, 38.4 mmol) in MIBK (20 mL) was injected into a glass packed bed flow reactor (2 mL) at rate of 0.1 mL/min. The reactor temperature was 55°C. The reaction mixture was collected and analyzed by HPLC resulting in 93% consumption of starting raw material and 95% Ester-PEG370. Example 5: Synthesis of Glucuronic Ester-PEG2000

[00207] In this embodiment, the reactor was charged with MIBK (2 mL), glucuronic acid (12 mg, 0.06 mmol) and PEG2000 (0.12 g, 0.06 mmol). The temperature was set to 55 °C, and the enzyme (4 mg) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The aqueous phase was extracted 2 times with ethyl acetate (50 mL). The combined organic phase was concentrated to dryness under vacuum (0.13 g, 75% yield, 100% glucuronic Ester-PEG2000).

Example 6: Synthesis of Ester-PEG6000

[00208] In this embodiment, the reactor was charged with MIBK (150 mL), PEG6000 (36 g, 6 mmol) and 3-methoxyphenylaceticacid (1 g, 6 mmol). The temperature was set to 50 °C, and the enzyme (16 mg) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The aqueous phase was extracted 2 times with ethyl acetate (50 mL). The combined organic phase was concentrated to dryness under vacuum (24 g, 65% yield, 100% Ester- PEG6000).

Example 7: Synthesis of Ester-PEG195

[00209] In this embodiment, the reactor was charged with MIBK (1 L), maleimidyl acetic acid (1 g, 6 mmol) and tetraethylene glycol (PEG4) (7.9 mL, 45 mmol). The temperature was set to 55 °C, and the enzyme (0.4 g) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and a 3% (w/w) NaCI aqueous solution previously adjusted to pH 1 was used to extract 3 times the organic phase. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum.

Example 8.1: Synthesis of PG-PEG195

[00210] In this embodiment, the reactor was charged with NaH (1.10 g, 40 mmol, 95%) and dissolved in cold THF (70 mL) under nitrogen. In an ice bath, and tetraethylene glycol (PEG4) (4equiv.) was dissolved in THF and added dropwise. After addition, the ice bath was removed and BnBr (1.8 g, 10 mmol, 99%) was added dropwise. The crude was stirred at 40° C for 24 hours until the reaction was complete, verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted with water (25 mL).

[00211] The aqueous layer was extracted with EtOAc (25 ml x 2). The combined organic phase was washed with water (25 ml x 4) and brine (25 ml x 2), dried over anhydrous NajSCU, and filtered. The filtrate was evaporated to dryness and further dried under vacuum giving the product BnO(PEG)4. The crude product was purified by gradient flash chromatography (Hexane/EtOAc 1:1). For each column 2 g or crude product was deposited on 10 g of silica. (Yield values: BnPEG4 (2.36 g, 79.4% yield, 100% BnPEG4).

Example 8.2: Synthesis of PG-PEG195-Ester

[00212] In this embodiment, the reactor was charged with diphenylmethane (100 mg, 0.59 mmol) and dissolved in THF (1 mL) under nitrogen. Ester-PEG195 (203 mg, 0.59 mmol) was added and triethylamine (180 mg, 1.78 mmol). The crude was stirred at G ’C for 24 hours until the reaction was complete, verified by HPLC. The reaction mixture was concentrated under vacuum.

(The reaction mixture was collected and analyzed by HPLC resulting in 41% consumption of starting raw material into Ester-PEG195-Bn).

Example 9: Synthesis of Cholesterol-Ester-PEG195

[00213] In this embodiment, the reactor was charged with MIBK (1 L), cholesterol (1 g, 2.6 mmol) and di(carboxylic acid) tetraethylene glycol (4.7 mL, 18 mmol). The temperature was set to 55 °C, and the enzyme (0.4 g) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and a 3%(w/w) NaCI aqueous solution previously adjusted to pH 1 was used to extract 3 times the organic phase. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum.

Example 10: Synthesis of triglyceride-Ester-PEG195

[00214] In this embodiment, the reactor was charged with MIBK (1 L), triglyceride (1 g, 2.6 mmol) and di(carboxylic acid) tetraethylene glycol (4.7 mL, 18 mmol). The temperature was set to 55 °C, and the enzyme (0.4 g) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and a 3% (w/w) NaCI aqueous solution previously adjusted to pH 1 was used to extract 3 times the organic phase. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum.

Example 11: Synthesis of ribose-Ester-PEG195

[00215] In this embodiment, the reactor was charged with MIBK (1 L), ribose (1 g, 6.7 mmol) and di(carboxylic acid) tetraethylene glycol (12.2 mL, 46.6 mmol). The temperature was set to 55 °C, and the enzyme (0.4 g) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and a 3% (w/w) NaCI aqueous solution previously adjusted to pH 1 was used to extract 3 times the organic phase. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum.

Example 12: Synthesis of Adenine-Ester-PEG195

[00216] In this embodiment, the reactor was charged with MIBK (1 L), adenine (1 g, 7.4 mmol) and di(carboxylic acid) tetraethylene glycol (14 mL, 52 mmol). The temperature was set to 55 °C, and the enzyme (0.4 g) was added manually to the reactor vessel. The reaction was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and a 3% (w/w) NaCI aqueous solution previously adjusted to pH 1 was used to extract 3 times the organic phase. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum.

Example 13: Synthesis of Ester-PEG195-Tosyl

[00217] In this embodiment, the reactor was charged with DCM (200 mL), Ester-PEG195 (6 g, 17.5 mmol), tosyl chloride (3.6 g, 19.3 mmol), and triethylamine (4.9 mL, 35 mmol). The temperature was set to 30 °C, and the mixture was stirred for 3 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (25 mL). The combined organic phase was concentrated to dryness under vacuum (8.7 g, 95% yield, 100% purity by HPLC). Example 14: Synthesis of Ester-PEG195-Mesyl

[00218] In this embodiment, the reactor was charged with DCM (15 mL), Ester-PEG195 (0.5 g, 1.5 mmol), methanosulfonyl chloride (0.2 g, 1.8 mmol), and triethylamine (0.4 mL, 2.9 mmol). The temperature was set to 30 °C, and the mixture was stirred for 3 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (10 mL). The combined organic phase was concentrated to dryness under vacuum (0.6 g, 93% yield, 99% purity by HPLC).

Example 15: Synthesis of Ester-PEG370-Ester

[00219] In this embodiment, the reactor was charged with THF (1 mL) and Ester-PEG195 (0.1 g, 0.29 mmol), and potassium bis(trimethylsilyl)amide (0.06 g, 0.29 mmol) at 0 °C. The mixture was stirred for 15 minutes. Ester-PEG195-Tosyl (0.14 g, 0.29 mmol) was added dropwise and after the temperature was set to 40 °C. The mixture was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (10 mL). The combined organic phase was concentrated to dryness under vacuum (0.4 g, 67% yield, 94% purity by HPLC).

Example 16: Synthesis of Ester-PEG370-PG

[00220] In this embodiment, the reactor was charged with THF (1 mL) and Bn-PEG195 (0.2 g, 0.7 mmol), and potassium bis(trimethylsilyl)amide (0.1 g, 0.7 mmol) at 0 °C. The mixture was stirred for 15 minutes. Ester-PEG195-Tosyl (0.87 g, 1.76 mmol) was added dropwise and after the temperature was set to 40 °C. The mixture was stirred for 20 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and a 3%(w/w) NaCI aqueous solution previously adjusted to pH 1 was used to extract 3 times the organic phase. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum.

Example 18: Synthesis of Ester-PEG722-Ester

[00221] In this embodiment, the reactor was charged with THF (1 mL) and Ester-PEG370 (0.1 g, 0.19 mmol), and potassium bis(trimethylsilyl)amide (0.04 g, 0.19 mmol) at 0 °C. The mixture was stirred for 15 minutes. Ester-PEG370-Tosyl (0.13 g, 0.19 mmol) was added dropwise and after the temperature was set to 40 °C. The mixture was stirred for 20 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and a 3%(w/w) NaCI aqueous solution previously adjusted to pH 1 was used to extract 3 times the organic phase. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum.

Example 19: Synthesis of Ester-PEG679-PG

[00222] In this embodiment, the reactor was charged with THF (1 mL) and Bn-PEG195 (0.2 g, 0.4 mmol), and potassium bis(trimethylsilyl)amide (0.08 g, 0.4 mmol) at 0 °C. The mixture was stirred for 15 minutes. Ester-PEG370-Tosyl (0.73 g, 0.4 mmol) was added dropwise and after the temperature was set to 40 °C. The mixture was stirred for 20 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and a 3%(w/w) NaCI aqueous solution previously adjusted to pH 1 was used to extract 3 times the organic phase. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum.

Example 20: Synthesis of Ester-PEG2000-Ester

[00223] In this embodiment, the reactor was charged with THF (1 mL) and Ester-PEGIOOO (1 g, 0.9 mmol), and potassium bis(trimethylsilyl)amide (0.2 g, 0.9 mmol) at 0 °C. The mixture was stirred for 15 minutes. Ester-PEGIOOO-Tosyl (1.1 g, 0.9 mmol) was added dropwise and after the temperature was set to 40 °C. The mixture was stirred for 20 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and a 3%(w/w) NaCI aqueous solution previously adjusted to pH 1 was used to extract 3 times the organic phase. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum.

Example 21: Synthesis of Ester-PEG2000-PG

[00224] In this embodiment, the reactor was charged with THF (1 mL) and Bn-PEGIOOO (1 g, 0.9 mmol), and potassium bis(trimethylsilyl)amide (0.18 g, 0.9 mmol) at 0 °C. The mixture was stirred for 15 minutes. Ester-PEGIOOO-Tosyl (3 g, 2.3 mmol) was added dropwise and after the temperature was set to 40 °C. The mixture was stirred for 20 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The organic phase was extracted 3 times with water (50 mL). The combined organic phase was concentrated to dryness under vacuum. The crude was dissolved in MTBE and a 3%(w/w) NaCI aqueous solution previously adjusted to pH 1 was used to extract 3 times the organic phase. Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum.

Example 22: Removal of Esters - PEG370

[00225] In this embodiment, the reactor was charged with water (10 mL) and the Ester-PEG370-Ester (1 g, 1.4 mmol). The temperature was set to 30 °C, and Lipase B from Candida Antarctica (5000 u/g, 100 mg) was added manually to the reactor vessel. The mixture was stirred for 6 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added lOmL MTBE. The aqueous phase was extracted 2 times with MTBE (30 mL). Then the aqueous phase was extracted with DCM and the organic phase was concentrated to dryness under vacuum (0.28g, 53% yield, 88% PEG370).

Example 23: Removal of Esters - Ester-PEG370

[00226] In this embodiment, the reactor was charged with water (3 mL) and DMSO (7 mL) and the Ester- PEG370-Ester (1 g, 1.4 mmol). The temperature was set to 25 °C, and Lipase B from Candida Antarctica (5000 u/g, 100 mg) was added manually to the reactor vessel. The mixture was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The aqueous phase was extracted 2 times with ethyl acetate (50 mL). The combined organic phase was concentrated to dryness under vacuum (0.59g, 74% yield, 85% Ester-PEG370).

Example 24: Removal of Ester - Bn- PEG370

[00227] In this embodiment, the reactor was charged with water (10 mL) and the Ester-PEG370-Ester (1 g, 1.4 mmol). The temperature was set to 30 °C, and the Lipase B from Candida Antarctica (5000 u/g, 100 mg) was added manually to the reactor vessel. The mixture was stirred for 24 hours until the reaction was complete verified by HPLC. The reaction mixture was concentrated under vacuum. To the crude was added water and ethyl acetate. The aqueous phase was extracted 2 times with ethyl acetate (20 mL). The combined organic phase was concentrated to dryness under vacuum.

Example 25: Deep eutectic solvents preparation general method

[00228] Hydrogen bond donor (HBD) and the hydrogen bond acceptor (HBA) described in table 5 were mixed in molar ratio range between 0.5 and 5 molar and stirred at temperature not higher than 60°C until the liquid obtained was uniform and clear. This procedure produces DES with melting point below 60°C preferentially below 52°C. [00229]Table 5: Experimental values for novel DES

[00230]Table 6 describes additional DES mixtures.

[00231]Table 6: Additional DES mixtures [00232] All new DES can be used to assist PEG purification by phase separation.

Example 26: Purification of PEG using DES previously prepared

[00233] A method for separating acid and PEG containing DES using a hydrophobic deep eutectic solvent based on that acid, comprising the steps of:

[00234] An aqueous solution containing lg of acid and 0.5 g of PEG370 was mixed with the hydrophobic deep eutectic solvent from Table 5-example 23.3 (3-methoxy phenyl acetic acid and thymol) in a molar ratio of 1:1, stirred at 10 °C for 30 minutes at and after standing for a while, the upper organic and aqueous phase were separated and analyzed by HPLC. The acid and PEG partitions were the following ones: Aqueous phase partitions: XA d=0.02, X_PEG4OO=1. Organic phase partitions: XA d=0.98, X_PEG4OO=O.

[00235]The PEG obtained has a purity equal or above 95%.

Example 27: Purification of PEG using DES prepared in situ

[00236] A method for separating acid and PEG containing DES using a hydrophobic deep eutectic solvent based on that acid, comprising the steps of:

[00237] An aqueous solutions containing lg of acid and 0.5 g of PEG370 were mixed with the hydrogen bound acceptor molar proportion described in Table 5 -example 23.3 (3-methoxy phenyl acetic acid and thymol) in a molar ratio of 1:1, stirred at 10 °C for 30 minutes at and after standing for a while, a droplet of organic phase was formed. The phases were separated and analyzed by HPLC. The acid and PEG partitions were the following ones: Aqueous phase partitions: XA d=0.03, X_PEG4OO=1. Organic phase partitions: x cid=0.97, X_PEG OO=O.

[00238]The PEG obtained has a purity equal or above 95%.

[00239] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range. [00240]The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[00241]The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The abovedescribed embodiments are combinable.

[00242]The following dependent claims further set out particular embodiments of the disclosure.

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Claims

C L A I M S

1. Method for obtaining an extended high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I'): formula (I) formula (I') comprising a step of enzymatic mono esterification, using a first enzyme in a first suitable organic solvent of a polyethylene glycol of general formula (II) with a carboxylic acid or carboxylic acid derivative of general formula (III), formula (II) formula (III) to obtain a mono esterified polyethylene glycol of general formula (IV), formula (IV) reacting the compound of general formula (IV) with a leaving group (LGi), in a second suitable organic solvent, to obtain the compound of general formula (V) formula (V) reacting the compound of general formula (IV) or a compound of general formula (IV') formula (IV') with the compound of general formula (V), in a third suitable organic solvent and in the presence of a first base, to obtain the compound of general formula (VI) or the compound of general formula VI', respectively, formula (VI') formula (VI) formula (VI') reacting the compound of general formula (VI) or the compound of general formula (VI'), with a second enzyme, in a suitable first aqueous-based or first water miscible solvent, to obtain the polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I'), respectively, wherein: the first enzyme and the second enzyme are independently selected from the group consisting of: lipase, esterase, protease, transferase, ligase or mixtures thereof;

Ri, Ri-, and R2 and independently selected;

R2 is selected from the group consisting of: hydrogen, cycloalkyl, C1-C12 substituted or nonsubstituted alkyl, C1-C12 substituted or non-substituted alkenyl, or C1-C12 substituted or nonsubstituted alkynyl; n and n' are independently selected; n and n' are at least 1.

2. Method according to any of the previous claims wherein the molecular weight of the high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') is at least 50 g/mol; preferably ranging from 150 g/mol - 20 000 g/mol; more preferably 195 g/mol - 10000 g/mol.

3. Method according to any of the previous claims wherein the molecular weight of the high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') ranges from 50 g/mol - 30 000 g/mol; preferably 150 g/mol - 20 000 g/mol; more preferably 195 g/mol - 10000 g/mol.

4. Method according to any of the previous claims wherein 2n or n+n' ranges from 2-200; preferably 5-100; more preferably 8-60.

5. Method according to any of the previous claims wherein n or n' ranges from 1-100; preferably 2-50; more preferably 4-30.

6. Method according to any of the previous claims wherein the concentration of the polyethylene glycol of general formula (II) in the first suitable organic solvent ranges from 0.005 mol/L to 50 mol/L; preferably 0.5 to 5 mol/L; more preferably 1 mol/L.

7. Method according to any of the previous claims wherein the step of reacting the compound of general formula (IV) with a leaving group (LGi) is performed at a temperature ranging from 25- 100°C; preferably 25-50 °C.

8. Method according to any of the previous claims wherein the step of reacting the compound of general formula (IV) or a compound of general formula (IV') with the compound of general formula (V) is performed at a temperature ranging from 24-120°C; preferably 30-50 °C.

9. Method according to any of the previous claims wherein LGi is selected from p-toluenesulfonate or methanesulfonate; preferably p-toluenesulfonate.

10. Method according to any of the previous claims wherein the step of reacting the compound of general formula (VI) or the compound of general formula (VI'), with a second enzyme, is performed at a temperature ranging from 5-100°C; preferably 25-50 °C.

11. Method according to any of the previous claims wherein R2 is selected from CH3, CH2CH3, CH2CH2CH3 or hydrogen; preferably CH3, CH2CH3 or hydrogen; more preferably CH3 or hydrogen.

12. Method according to the previous claim wherein R2 is hydrogen.

13. Method according to any of the previous claims wherein Ri and/or Ri- are a non-substituted alkyl, a non-substituted alkenyl, a non-substituted alkynyl, a non-substituted cycloalkyl, a non-substituted heterocycloalkyl, a non-substituted aryl or a non-substituted heteroaryl.

14. Method according to any of the previous claims wherein Ri and/or Ri- are a substituted or nonsubstituted phenyl, a substituted or non-substituted naphthalenyl, a substituted or non-substituted tetrahydronephthalenyl or a substituted or non-substituted indane; preferably a non-substituted phenyl, a non-substituted naphthalenyl, a non-substituted tetrahydronephthalenyl or a nonsubstituted indane.

15. Method according to any of the previous claims wherein Ri and/or Ri- are a substituted or nonsubstituted monocyclic aromatic ring selected from the list consisting of: furyl, furazanyl, imidazolyl, isothiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyranyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, pyridyl, thienyl, thiazolyl, triazolyl, tetrazolyl, triazinyl, tetrazinyl; preferably a non-substituted monocyclic aromatic ring selected from the list consisting of: furyl, furazanyl, imidazolyl, isothiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyranyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl, pyridyl, thienyl, thiazolyl, triazolyl, tetrazolyl, triazinyl, tetrazinyl.

16. Method according to any of the previous claims wherein Ri and/or Ri- are a substituted or nonsubstituted bicyclic aromatic ring selected from the group consisting of: azaindolyl, benzothienyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzoxadiazolyl, benzothiadiazolyl, benzofuranyl, cinnolinyl, furopyridyl, imidazopyridyl, indolyl, isoindolyl, isobenzofuranyl, indolizinyl, indazolyl, isoquinolinyl, naphthyridinyl, quinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pteridinyl, purinyl, pyrrolopyridyl; preferably Ri is a non-substituted bicyclic aromatic ring selected from the group consisting of: azaindolyl, benzothienyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzoxadiazolyl, benzothiadiazolyl, benzofuranyl, cinnolinyl, furopyridyl, imidazopyridyl, indolyl, isoindolyl, isobenzofuranyl, indolizinyl, indazolyl, isoquinolinyl, naphthyridinyl, quinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pteridinyl, purinyl, pyrrolopyridyl.

17. Method according to any of the previous claims wherein the carboxylic acid or carboxylic acid derivative is selected from the group consisting of: benzoic acid, amino-substituted phenylacetic acid, nitro-substituted phenylacetic acid, nitro-substituted benzoic acid, methoxy-substituted phenylacetic acid, methyl-substituted phenylacetic acid, 3-methoxycinnamic acid, 2-(tiophen-3- yl)acetic acid, (E)-5-methoxypent-4-enoic acid, 2-(furan-3-yl)acetic acid, 2-(quinolin-3-yl)acetic acid, 2-(3-methoxyphenyl)-2-oxoacetic acid, 3-butynoic acid, maleimidyl acetic acid, pyridin-3-yl acetic acid, pyridin-4-yl acetic acid, maleimidyl propionic acid, maleic anhydride, 3- acetamidophenylacetic acid, 3-methoxycinnamic acid, methyl-(3-methoxy)cinnamate, triphenyl acetic acid, diphenyl acetic acid, hydroxyl-substituted phenylacetic acid, phenyl acetic acid, (3- methoxy) phenylacetate, triglycerides, diglycerides, monoglycerides, glucuronic acid, isosaccharinic acid, gluconic acid or glucaric acid, or esters thereof.

18. Method according to any of the previous claims wherein the carboxylic acid or carboxylic acid derivative is selected from the list consisting of: 3-aminophenylacetic acid, 4-aminophenylacetic acid, 2-aminophenylacetic acid, 3-nitrophenylacetic acid, 4-nitrophenylacetic acid, 2- nitrophenylacetic acid, 3- nitrobenzoic acid, 4- nitrobenzoic acid, 2-nitrobenzoic acid, 3- methoxyphenylacetic acid, 4-methoxyphenylacetic acid, 2-methoxyphenylacetic acid, 3- methylphenylacetic acid, 4-methylphenylacetic acid, 2-methylphenylacetic acid, 3- methoxycinnamic acid, 2-(tiophen-3-yl)acetic acid, (E)-5-methoxypent-4-enoic acid, 2-(furan-3- yl)acetic acid, 2-(quinolin-3-yl)acetic acid, 2-(3-methoxyphenyl)-2-oxoacetic acid, 3-butynoic acid, maleimidyl acetic acid, pyridin-3-yl acetic acid, pyridin-4-yl acetic acid, maleimidyl propanoic acid, maleic anhydride, 3-acetamidophenylacetic acid, 3-methoxycinnamic acid, methyl-(3- methoxy)cinnamate, triphenyl acetic acid, diphenyl acetic acid, 3-hydroxy phenylacetic acid, 4- hydroxy phenylacetic acid, 2-hydroxy phenylacetic acid, phenyl acetic acid, (3-methoxy) phenylacetate, glucuronic acid, isosaccharinic acid, gluconic acid ,glucaric acid, or esters thereof.

19. Method according to any of the previous claims wherein the carboxylic acid or carboxylic acid derivative is selected from the list consisting of: 2-(m-tolyl)acetic acid, 3-methoxyphenylacetic acid, (3-methoxy) phenylacetate, methyl-3-methoxy cinnamate, glucuronic acid and maleimidyl acetic acid; preferably 3-methoxyphenyl acetic acid or (3-methoxy) phenylacetate.

20. Method according to any of the previous claims wherein the carboxylic acid or carboxylic acid derivative is selected from the group consisting of: alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, ornithine, citrulline, diaminobutyric acid, p-alanine, y-aminobutyric acid, hydroxylysine, hydroxyproline, desmosine, or isodesmosine, or esters thereof.

21. Method according to any of the previous claims wherein the carboxylic acid or carboxylic acid derivative is selected from the group consisting of: 3-methyoxyphenylacetic acid; 3- methylphenylacetic acid; phenylacetic acid; 3-aminophenylacetic acid; 4-aminophenylacetic acid; 3- hydroxyphenylacetic acid; Benzoic acid; 3-acetaminophenylacetic acid; 4-nitrophenylacetic acid; Triphenylacetic acid; 2-methoxyphenylacetic acid, or esters thereof.

22. Method according to any of the previous claims wherein the amount of the first enzyme is at least 1 mg for each mmol of polyethylene glycol of general formula (II); preferably the amount of the first enzyme ranges from 1 mg to 150 mg for each mmol of polyethylene glycol of general formula (II); more preferably the amount of the first enzyme ranges from 1 mg to 30 mg for each mmol of polyethylene glycol of general formula (II); even more preferably the amount of the first enzyme ranges from 2 mg to 15 mg for each mmol of polyethylene glycol of general formula (II); even more preferably the amount of the first enzyme ranges from 3 mg to 13 mg for each mmol of polyethylene glycol of general formula (II).

23. Method according to any of the previous claims wherein the amount of the second enzyme is at least 1 mg for each mmol of compound of general formula (VI) or compound of general formula (VI'); preferably the amount of the second enzyme ranges from 1 mg to 150 mg for each mmol of compound of general formula (VI) or compound of general formula (VI'); more preferably the amount of the second enzyme ranges from 1 mg to 30 mg for each mmol of compound of general formula (VI) or compound of general formula (VI'); even more preferably the amount of the second enzyme ranges from 2 mg to 15 mg for each mmol of compound of general formula (VI) or compound of general formula (VI'); even more preferably the amount of the second enzyme ranges from 3 mg to 13 mg for each mmol of compound of general formula (VI) or compound of general formula (VI').

24. Method according to any of the previous claims wherein the enzymatic mono esterification is performed at a temperature ranging from 0°C - 120°C; preferably 40°C -70°C; more preferably 50°C -60°C; even more preferably 55°C.

25. Method according to any of the previous claims wherein the enzymatic mono esterification is performed in a pH ranging from 1 to 8; preferably 2-6.

26. Method according to any of the previous claims wherein the first suitable organic solvent is selected from the group consisting of: heptane, ketone, methyl isobutyl ketone, acetonitrile, tetrahydrofuran, toluene, tert-amyl alcohol, ethyl acetate, diethyl ether, dioxane, dimethylsulfoxide, cyclohexanone, methyl ethyl ketone, isopropyl alcohol, Methyl tertbutyl alcohol, diisopropylalcohol, methyltetrahydrofuran, anisole, or mixtures thereof.

27. Method according to any of the previous claims wherein the first suitable organic solvent is methyl isobutyl ketone.

28. Method according to any of the previous claims further comprising the step of: reacting the compound of general formula (V) with a compound of general formula (VIII),

29. Method according to any of the previous claim further comprising the step of: reacting the compound of general formula IX with a third enzyme, in a suitable second aqueousbased or second water miscible solvent, to obtain the compound of general formula (XV) formula (XV) wherein: the third enzyme is selected from the group consisting of: lipase, esterase, protease, transferase, ligase or mixtures thereof; the reaction is performed at a temperature ranging from 5-100°C.

30. Method according to the previous claim wherein the amount of the third enzyme is at least 1 mg for each mmol of compound of general formula IX ; preferably the amount of enzyme ranges from 1 mg to 150 mg for each mmol of compound of general formula IX; more preferably the amount of enzyme ranges from 1 mg to 30 mg for each mmol of compound of general formula IX; even more preferably the amount of enzyme ranges from 2 mg to 15 mg for each mmol of compound of general formula IX ; even more preferably the amount of enzyme ranges from 3 mg to 13 mg for each mmol of compound of general formula IX .

31. Method according to any of the previous claims 28-30 wherein the step is performed in a pH ranging from 1 to 8; preferably 2-6.

32. Method according to any of the previous claims wherein the first suitable aqueous-based or first water miscible solvent and the second suitable aqueous-based or second water miscible solvent are independently selected from the group consisting of: water, ethanol, methanol, acetone, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, or mixtures thereof; preferably water.

33. Method according to the previous claim wherein the first suitable aqueous-based or first water miscible solvent and the second suitable aqueous-based or second water miscible solvent is water.

34. Method according to any of the previous claims wherein the activity of the first enzyme or the activity of the second enzyme or the activity of the third enzyme ranges from 100 U/g to 20000 U/g; preferably 200 U/g to 15000 U/g; more preferably 250 U/g to 10000 U/g.

35. Method according to any of the previous claims wherein the first, the second and/or the third enzyme are independently selected from a lipase, esterase, protease, transferase or ligase obtained from a microorganism selected from the list consisting of: Alcaligenes spp, Aspergillus spp, Candida rugosa , Saccharomyces cerevisiae, Candida Antarctica, Chromobacterium spp, Rhizomucor spp, Penicilium spp, Pseudomonas spp, Rhizopus spp, Thermomyces spp, Geotrichum spp, Mucor spp, Burkholderia spp , Alcaligenes spp, Candida spp, Chromobacterium spp, , Bacillus subtilis, Rhizopus spp, Serratia marcescens, Escherichia coli, Pseudomonas fluorescens, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris, Schizosaccharomyces pombe, Pichia pastoris, Kluyveromyces lactis, Streptomyces spp, Bacillus cereus, Salmonella typhimurium, , Streptomyces spp, Staphylococcus spp, Pichia spp, Saccharomyces spp, Schizosaccharomyces spp, Schwanniomyces spp, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe or Fusarium solani pisi, or mixtures thereof.

36. Method according to any of the previous claims wherein the first, the second and/or the third enzyme are independently selected a lipase, esterase or protease obtained from a microorganism selected from the list consisting of: Candida spp, Thermomyses lanuginosus and Mucor miehei.

37. Method according to any of the previous claims wherein the first, the second and/or the third enzyme is a lipase (triacylglycerol acyl hydrolases, EC number 3.1.1.3).

38. Method according to any of the previous claims wherein the first, the second and/ or the third enzyme is lipase enzyme obtained from Candida antarctica B; preferably Candida antarctica lipase B (CALB) immobilized on a hydrophobic carrier; more preferably Candida antarctica lipase B (CALB) immobilized on acrylic resin.

39. Method according to any of the previous claims wherein the first, the second and/or the third enzyme is lipase from Mucor miehei; preferably lipase from Mucor miehei immobilized on a hydrophobic carrier.

40. Method according to the previous claim 29 further comprising the step of: removing the protecting group (PG) from the compound of general formula (XV), in a fifth suitable organic solvent, to obtain a compound of general formula (I")

Formula (I").

41. Method according to the previous claim wherein the step of removing the protecting group (PG) is performed with palladium on carbon (Pd/C), preferably 5-10% Pd by weight (of the compound of formula (XV), under hydrogen atmosphere at temperature ranging from 25-50°C and pressure ranging from 1-30 bar.

42. Method according to any of the previous claims further comprising the step of: reacting the polyethylene glycol of general formula (I), polyethylene glycol of general formula (I') or polyethylene glycol of general formula (II'), with a leaving group (LG2), in a fifth suitable organic solvent, to obtain the compound of general formula (XII), compound of general formula (XII') or compound of general formula (XII"), respectively formula (XII) formula (XII') formula (XII") wherein:

43. Method according to the previous claim further comprising the step of: reacting a compound of general formula (XII), a compound of general formula (XII') or a compound of general formula (XII") with a compound of general formula (IV), in a sixth suitable organic solvent and in the presence of a third base, to obtain the compound of general formula (XIII), compound of general formula (XIII') or compound of general formula (XIII"), respectively, formula (XIII) formula (XIII') formula (XIII") wherein the reaction is performed at a temperature ranging from 24-120°C.

44. Method according to any of the previous claims wherein the second, third, fourth, fifth or sixth suitable organic solvent are independently selected from the group consisting of: dichloromethane, tetrahydrofuran, diethyl ether, Isopropylether, 2-methyl tetrahydrofuran, tetrahydrofuran, Dioxane, Methyl tert-Butyl Ether, Ethylene Glycol Dimethyl Ether, Ethyl Acetate, Methyl Acetate, Butyl Acetate, Isopropyl Acetate, Isopropyl Acetate, Acetone, Methyl Ethyl Ketone, Cyclohexanone, Methyl Isobutyl Ketone, Diisobutyl Ketone, isopropyl alcohol, Methyl tertbutyl alcohol, diisopropylalcohol, methyltetrahydrofuran, anisole; preferably selected from dichloromethane, tetrahydrofuran, diethyl ether, Isopropylether, 2-methyl tetrahydrofuran, tetrahydrofuran, or mixtures thereof.

45. Method according to any of the previous claims wherein the second suitable organic solvent, the third suitable organic solvent, the fourth suitable organic solvent, the fifth suitable organic solvent and the sixth suitable organic solvent is tetrahydrofuran.

46. Method according to any of the previous claims wherein the first base, the second base and the third base are independently selected from the list consisting of: sodium carbonate (NajCOs), Potassium carbonate (K2CO3), Sodium bicarbonate (NaHCOs), Potassium bicarbonate (KHCO3), Sodium methoxide (NaOCHs), Sodium ethoxide (NaOEt), Potassium tert-butoxide (KOtBu), Lithium diisopropylamide (LDA), Triethylamine (EtsN), Diisopropylethylamine (DIPEA), Pyridine, Sodium hydride (NaH), Potassium hydride (KH), l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), potassium bis(trimethylsilyl)amide; or mixtures thereof.

47. Method according to any of the previous claims wherein the first base, the second base and the third base is potassium bis(trimethylsilyl)amide.

48. Method according to any of the previous claims wherein the method is performed at a pressure at or above atmosphere pressure; preferably equal or above 101.32500 kPa (1 atmosphere).

49. Method according to any of the previous claims further comprising a step of purifying the polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I').

50. Method according to the previous claim wherein the purification step comprises the following steps: providing an aqueous mixture comprising a suitable aqueous-based or water miscible solvent, the polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I'), and one or more impurities; contacting the aqueous mixture with a deep eutectic solvent comprising a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA), wherein the deep eutectic solvent selectively dissolves said impurities; separating the aqueous phase comprising the purified polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I'); and optionally recovering the purified polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I') by precipitation, filtration, or evaporation.

51. Stable high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') obtainable by the method described in any of the previous claim.

52. Stable high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') according to the previous claim wherein the polyethylene glycol is selected from the list consisting of: PEG195, PEG370, PEG679, PEG722, PEG2000, PEG6000.

53. Stable high molecular weight monodispersed polyethylene glycol according to any of the previous claims 51-52wherein the polydispersity index (PDI) is about 1.00; preferably ranges from 1.00 to 1.10; more preferably 1.00 to 1.08.

54. Stable high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') according to any of the previous claims 51-53 for use in medicine, preferably as a pharmaceutical acceptable excipient.

55. Stable high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') according to any of the previous claims 51-53 for use in medicinal devices, preferably as lubricant for medical devices, or coatings for implants.

56. Use of the stable high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I') according to any of the previous claims 51-53 as a cosmetic acceptable excipient for cosmetical formulations or devices.

57. Compound of formula IV or formula IV' wherein the compound is selected from the list consisting of:

58. Deep eutectic solvent for purifying polyethylene glycol comprising: a mixture of a carboxylic acid and a hydrogen bond acceptor in a molar ratio ranging from 1:0.2 to 1:5, wherein: the carboxylic acid is selected from the list consisting of: 3-aminophenylacetic acid, 4- aminophenylacetic acid, 2-aminophenylacetic acid, 3-nitrophenylacetic acid, 4-nitrophenylacetic acid, 2-nitrophenylacetic acid, 3- nitrobenzoic acid, 4- nitrobenzoic acid, 2-nitrobenzoic acid, 3- methoxyphenylacetic acid, 4-methoxyphenylacetic acid, 2-methoxyphenylacetic acid, 3- methylphenylacetic acid, 4-methylphenylacetic acid, 2-methylphenylacetic acid, 3- methoxycinnamic acid, 2-(tiophen-3-yl)acetic acid, (E)-5-methoxypent-4-enoic acid, 2-(furan-3- yl)acetic acid, 2-(quinolin-3-yl)acetic acid, 2-(3-methoxyphenyl)-2-oxoacetic acid, 3-butynoic acid, maleimidyl acetic acid, pyridin-3-yl acetic acid, pyridin-4-yl acetic acid, maleimidyl propionic acid, 3- acetamidophenylacetic acid, 3-methoxycinnamic acid, methyl-(3-methoxy)cinnamate, triphenyl acetic acid, diphenyl acetic acid, 3-hydroxy phenylacetic acid, 4-hydroxy phenylacetic acid, 2- hydroxy phenylacetic acid, phenyl acetic acid, glucuronic acid, isosaccharinic acid, gluconic acid or glucaric acid; the hydrogen bond acceptor is selected from the group consisting of: menthol, terpineol, geraniol, linalol, methyltriphenylphosphonium, thymol, limonene, terpinene, eugenol, hinokitiol, carvone, pirene, coumarin, 6-methyl-coumarin, lidocaine, nicotinic acid, humulene, taxadiene, geosmin, ocimene, nerolidol, farnesol, tetra ammonium chloride, proline, alanine, choline, or mixtures thereof.

59. Deep eutectic solvent according to the previous claim wherein the carboxylic acid is selected from the list consisting of: 3-aminophenylacetic acid, 4-aminophenylacetic acid, 2-aminophenylacetic acid, 3-nitrophenylacetic acid, 4-nitrophenylacetic acid, 2-nitrophenylacetic acid, 3- nitrobenzoic acid, 4- nitrobenzoic acid, 2-nitrobenzoic acid, 3-methoxyphenylacetic acid, 4-methoxyphenylacetic acid, 2-methoxyphenylacetic acid, 3-methylphenylacetic acid, 4-methylphenylacetic acid, 2- methylphenylacetic acid, 3-methoxycinnamic acid, 2-(tiophen-3-yl)acetic acid, (E)-5-methoxypent- 4-enoic acid, 2-(furan-3-yl)acetic acid, 2-(quinolin-3-yl)acetic acid, 2-(3-methoxyphenyl)-2-oxoacetic acid, 3-butynoic acid, maleimidyl acetic acid, pyridin-3-yl acetic acid, pyridin-4-yl acetic acid, maleimidyl propionic acid, 3-acetamidophenylacetic acid, 3-methoxycinnamic acid, methyl-(3- methoxy)cinnamate, triphenyl acetic acid, diphenyl acetic acid, 3-hydroxy phenylacetic acid, 4- hydroxy phenylacetic acid, 2-hydroxy phenylacetic acid, phenyl acetic acid, glucuronic acid, isosaccharinic acid, gluconic acid or glucaric acid.

60. Deep eutectic solvent according to the previous claim wherein the carboxylic acid is selected from the list consisting of: 2-(m-tolyl)acetic acid, 3-methoxyphenylacetic acid, methyl-3-methoxy cinnamate, glucuronic acid or maleimidyl acetic acid.

61. Deep eutectic solvent according to the previous claim wherein the carboxylic acid is 3- methoxyphenyl acetic acid.

62. Deep eutectic solvent according to any of the previous claims 58-61 wherein the hydrogen bond acceptor is selected from the list consisting of: coumarin, menthol, thymol, choline chloride, 6- methyl coumarin or tetrabutylammonium chloride.

63. Deep eutectic solvent according to any of the previous claims 58-62 wherein the hydrogen bond acceptor is thymol.

64. Deep eutectic solvent according to any of the previous claims 58-63 wherein the molar ratio of the carboxylic acid and the hydrogen bond acceptor ranges from 1:0.2 to 1:5; preferably 1:0.5 to 1:2; more preferably 1:1.

65. Deep eutectic solvent according to any of the previous claims 58-64 comprising a mixture of 3- methoxy phenylacetic acid and thymol in a molar ratio ranging from 1:0.2 to 1:5; preferably 1:0.5 to 1:2; more preferably 1:1.

66. Deep eutectic solvent according to any of the previous claims 58-65 comprising a mixture of 3- methoxy phenylacetic acid and thymol in a molar ratio of 1:1.

67. Deep eutectic solvent according to any of the previous claims 58-66 comprising: a mixture of 3- methoxy phenylacetic acid and coumarin in a molar ratio of 1:1; or a mixture of 3-methoxy phenyl acetic acid and menthol in a molar ratio of 1:3; or a mixture of 3-methoxy phenyl acetic and thymol in a molar ratio of 1:1; or a mixture of 3-methoxy phenyl acetic and choline chloride in a molar ratio of 3:1; or a mixture of 3-methoxy phenyl acetic and 6-methyl coumarin in a molar ratio of 1:1; or a mixture of 3-methoxy phenyl acetic and tetrabutylammonium chloride in a molar ratio of 1:1.

68. Deep eutectic solvent according to any of the previous claims 58-67 comprising: a mixture of 3- methoxy phenylacetic acid and lidocaine in a molar ratio of 1:1; or a mixture of 3-methoxy phenyl acetic acid and methyl- triphenylphosphonium bromide in a molar ratio of 1:2.2; or a mixture of 3- methyl phenyl acetic acid and lidocaine in a molar ratio of 1:1.5; or a mixture of 3-methyl phenyl acetic acid and menthol in a molar ratio of 1:1.2; or a mixture of 3-methyl phenyl acetic acid and thymol in a molar ratio of 1:1; or a mixture of 3-methyl phenyl acetic acid and coumarin in a molar ratio of 1:2; or a mixture of 3-aminophenyl acetic acid and choline chloride in a molar ratio of 1:1.3; or a mixture of 3-aminophenyl acetic acid and thymol in a molar ratio of 1:1; or a mixture of 3- aminophenyl acetic acid and 6-methylcoumarin in a molar ratio of 1:1.4; or a mixture of 3- aminophenyl acetic acid and coumarin in a molar ratio of 1:1.4; or a mixture of 3-aminophenyl acetic acid and lidocaine in a molar ratio of 1:1.5; or a mixture of phenyl acetic acid and Thymol in a molar ratio of 1:1.2; or a mixture of phenyl acetic acid and Menthol in a molar ratio of 1:1.2; or a mixture of phenyl acetic acid and Coumarin in a molar ratio of 1:1.2; or a mixture of phenyl acetic acid and 6-methylcoumarin in a molar ratio of 1:1.2; or a mixture of phenyl acetic acid and Lidocaine in a molar ratio of 1:1.

69. Use of the deep eutectic solvent according to any of the previous claims 58-68 as a purifier of mono dispersive polyethylene glycol; preferably a high molecular weight monodispersed polyethylene glycol of general formula (I) or general formula (I').

70. Use of the deep eutectic solvent according to any of the previous claims 58-69 as a carboxylic acid extractor, preferably a carboxylic acid of general formula III.

71. Use of the deep eutectic solvent according to any of the previous claims 58-70 for extracting a carboxylic acid selected from the group consisting of: 3-aminophenylacetic acid, 4- aminophenylacetic acid, 2-aminophenylacetic acid, 3-nitrophenylacetic acid, 4-nitrophenylacetic acid, 2-nitrophenylacetic acid, 3- nitrobenzoic acid, 4- nitrobenzoic acid, 2-nitrobenzoic acid, 3- methoxyphenylacetic acid, 4-methoxyphenylacetic acid, 2-methoxyphenylacetic acid, 3- methylphenylacetic acid, 4-methylphenylacetic acid, 2-methylphenylacetic acid, 3- methoxycinnamic acid, 2-(tiophen-3-yl)acetic acid, (E)-5-methoxypent-4-enoic acid, 2-(furan-3- yl)acetic acid, 2-(quinolin-3-yl)acetic acid, 2-(3-methoxyphenyl)-2-oxoacetic acid, 3-butynoic acid, maleimidyl acetic acid, pyridin-3-yl acetic acid, pyridin-4-yl acetic acid, maleimidyl propanoic acid, 3-acetamidophenylacetic acid, 3-methoxycinnamic acid, methyl-(3-methoxy)cinnamate, triphenyl acetic acid, diphenyl acetic acid, 3-hydroxy phenylacetic acid, 4-hydroxy phenylacetic acid, 2- hydroxy phenylacetic acid, phenyl acetic acid, glucuronic acid, isosaccharinic acid, gluconic acid or glucaric acid; preferably 2-(m-tolyl)acetic acid, 3-methoxyphenylacetic acid, methyl-3-methoxy cinnamate, glucuronic acid and maleimidyl acetic acid; more preferably 3-methoxyphenyl acetic acid.

72. Method according to any of the previous claims 1-48 further comprising the step of: adding the deep eutectic solvent according to any of the previous claims 58-68 to the aqueous mixture comprising the suitable aqueous-based or water miscible solvent, the polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I'), and one or more impurities; obtain a first liquid phase comprising the aqueous solvent and the polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I') and a second liquid phase comprising the deep eutectic solvent and one or more impurities; separating and collecting the aqueous phase comprising the polyethylene glycol of general formula (I) or the polyethylene glycol of general formula (I').