CN111902430A

CN111902430A - Column [5] arenes for attachment peptides for highly permeable and selective artificial water channels and their synthesis and characterization

Info

Publication number: CN111902430A
Application number: CN201980020906.0A
Authority: CN
Inventors: 李庆; 王蓉; 陈俊丰
Original assignee: Nanyang Technological University
Current assignee: Nanyang Technological University
Priority date: 2018-03-21
Filing date: 2019-03-21
Publication date: 2020-11-06
Anticipated expiration: 2039-03-21
Also published as: CN111902430B; SG11202004694YA; WO2019182517A1

Abstract

According to the present invention, a method for synthesizing and isolating (pS) -pillared [5] arenes and (pR) -pillared [5] arenes for attachment of peptides is provided. (pR) -pillared [5] arenes attached to peptides can form single molecule channels with nanotube structures for water transport. A method for synthesizing (pR) -column [5] arene attached peptide for developing high permeability and selective water channels is discussed, which includes incorporating the (pR) -column [5] arene attached peptide into a liposome membrane, measuring water permeability of the (pR) -column [5] arene attached peptide by stop flow light scattering experiments, determining single channel water permeability by circular dichroism and UV-vis techniques, calculating activation energy by measuring water permeability at different temperatures, and estimating relative solute retention by using different solutes as osmotic agents.

Description

Column [5] arenes for attachment peptides for highly permeable and selective artificial water channels and their synthesis and characterization

Cross Reference to Related Applications

This application claims priority to singapore patent application 10201802298R filed on 3/21/2018, the contents of which are incorporated herein by reference in their entirety for all purposes.

Technical Field

The invention relates to a method for synthesizing column [5] arene hydrolysate of attached peptide. The invention also relates to such hydrolysates and their use.

Background

Aquaporin (AQP) is formed by the narrowest constriction

The hourglass structure of (a) constitutes aquaporins. This unique structure not only allows for rapid water transport (about 10)⁸-10⁹Molecules/sec/channel) and also prevents solute transport. This transport property has stimulated extensive research into the modification of AQPs into synthetic membranes for desalination and water purification applications. Since then, several classes of AQP-based biomimetic membranes with excellent water flux and desalination performance have been designed and manufactured. However, the high AQP production cost, low AQP stability and challenges in AQP film manufacturing have hindered the large scale application of AQP.

Therefore, artificial water channels compatibly incorporated into lipid membranes, configured with a water permeable central pore and an outer hydrophobic shell, are considered as alternatives to AQPs. Several types of artificial water channels have been designed and developed based on chemical synthesis and self-assembly to improve the water guiding rate and selectivity of the artificial water channels. However, conventional water channels do not achieve the water permeability and/or selectivity of AQP.

Moreover, the exploration of the artificial water channel is still in the concept verification stage and is far from the practical application.

Accordingly, there is a need to provide a solution that ameliorates one or more of the limitations described above. The solution should at least provide a compound that can be configured as an artificial water channel with high water permeability and improved selective transport properties.

Disclosure of Invention

In one aspect, there is provided a method of synthesizing a hydrolysate of column [5] arene of an attachment peptide, the method comprising:

formation of a column [5] arene substituted with a carboxyl group from a dialkoxybenzene;

column substituted with carboxyl groups [5]Arene and tripeptide are mixed in organic solvent to form columns for attachment of peptides [5]Aromatic hydrocarbons, wherein the tripeptide has a terminal-NH₂Groups and terminal esters; and

hydrolyzing the peptide-attached column [5] arene in the presence of a base to obtain a peptide-attached column [5] arene hydrolysate.

In another aspect, there is provided a hydrolysis product of a peptide-attached column [5] arene configured as a water channel in a liposome membrane, wherein the hydrolysis product of the peptide-attached column [5] arene is represented by the formula:

wherein R represents a tripeptide having a terminal-NH-group and a terminal-COOH group, wherein the terminal-NH-group forms part of an amide bond, and wherein the terminal-COOH group extends away from the amide bond.

In another aspect, there is provided a method of synthesizing a liposomal membrane comprising a hydrolysate of column [5] arene attached to a peptide, the method comprising:

synthesizing a hydrolysate of column [5] arene attached to the peptide according to the method described in the various embodiments of the first aspect;

mixing the hydrolysate with a plurality of lipids to form a mixture;

forming a liposome membrane from the mixture;

contacting the liposome membrane with a buffer to form a suspension comprising the hydrolysate and a plurality of lipids; and

the suspension is extruded through a membrane to form a liposomal membrane.

In another aspect, there is provided a liposomal membrane comprising a plurality of lipids and at least one hydrolysis product of a peptide-attached column [5] arene, wherein the at least one hydrolysis product of a peptide-attached column [5] arene is represented by the formula:

wherein R represents a tripeptide having a terminal-NH-group and a terminal-COOH group, wherein the terminal-NH-group forms an amide bond with the carbonyl carbon, and wherein the terminal-COOH group extends away from the amide bond.

In another aspect, there is provided a method of determining the insertion efficiency of a hydrolysate of column [5] arene attached to a peptide obtained according to the method described in the various embodiments of the first aspect, the method comprising:

mixing the liposome membrane obtained according to the method described herein with a buffer solution;

subjecting the buffer solution to a 0 UV-visible spectrum having a UV-visible absorption wavelength of 293nm to detect UV-visible absorbance from the pi-pi transition of benzene; and

the UV-visible absorbance was correlated with a UV-visible absorbance-concentration standard curve to determine the intercalation efficiency.

subjecting the buffer solution to a circular dichroism spectrum having a circular dichroism absorption wavelength of 306nm to detect circular dichroism absorbance from pi-pi transition of benzene; and

circular dichroism absorbance was correlated with a circular dichroism absorbance-concentration standard curve to determine the intercalation efficiency.

Drawings

The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention. In the following description, various embodiments of the present invention will be described with reference to the following drawings, in which:

FIG. 1 shows the synthesis of (pS) -pillared [5] arenes attached to peptides and (pR) -pillared [5] arenes attached to peptides (pS-PH and pR-PH);

FIG. 2A shows the (pS) -and (pR) -isomers of planar chiral column [5] arenes;

FIG. 2B shows the chemical structures of the peptide-attached (pS) -column [5] arene and the peptide-attached (pR) -column [5] arene (i.e., pS-PM and pR-PM, respectively) and their corresponding hydrolysates (pS-PH and pR-PH, respectively);

FIG. 2C shows DMSO-d at 298K₆Fractions of pS-PM, pR-PM and mixtures of pS-PM and pR-PM¹HNMR spectroscopy;

FIG. 2D shows Circular Dichroism (CD) spectra of pS-PM, pR-PM, pS-PH and pR-PH in Tetrahydrofuran (THF) at 298K;

FIG. 2E shows UV-vis spectra of pS-PM, pR-PM, pS-PH and pR-PH in THF at 298K;

FIG. 3 shows the reaction in DMSO-d₆Of middle pS-PM¹H NMR spectrum;

FIG. 4 shows the reaction in DMSO-d₆Of middle pS-PM¹³C NMR spectrum;

FIG. 5 shows a MALDI-TOF (matrix assisted laser Desorption/ionization time of flight mass Spectrometry) mass spectrum of pS-PM;

FIG. 6 shows the reaction in DMSO-d₆Of pR-PM¹H NMR spectrum;

FIG. 7 shows the reaction in DMSO-d₆Of pR-PM¹³C NMR spectrum;

FIG. 8 shows a MALDI-TOF mass spectrum of pR-PM;

FIG. 9 shows the reaction in DMSO-d₆Of medium pS-PH¹H NMR spectrum;

FIG. 10 shows the reaction in DMSO-d₆Of medium pS-PH¹³C NMR spectrum;

FIG. 11 shows a MALDI-TOF mass spectrum of pS-PM;

FIG. 12 shows the reaction in DMSO-d₆Of pR-PH¹H NMR spectrum;

FIG. 13 shows the reaction in DMSO-d₆Of pR-PH¹³C NMR lightA spectrum;

FIG. 14 shows MALDI-TOF mass spectrum of pR-PH;

FIG. 15A is a schematic of a stopped-flow light scattering test (stopped-flow light scattering test) used for shrinkage experiments;

FIG. 15B shows the stopped flow light scattering curve for liposomes containing pS-PH channels and having different Channel-to-lipid molar ratios (CLRs) after exposure to 400mM sucrose in a hypertonic solution at 10 ℃;

FIG. 15C shows stopped flow light scattering curves for liposomes containing pR-PH channels and having different channel-to-lipid mole ratios (CLR) after exposure to 400mM sucrose in a hypertonic solution at 10 ℃;

FIG. 15D shows the net water permeability of pR-PH in liposomes with different channel-to-lipid molar ratios (CLR) measured at 10 ℃ under hypertonic conditions;

FIG. 15E shows an Arrhenius relationship chart for calculating activation energy;

FIG. 15F is a schematic of a solubilized pR-PH channel in a buffered solution containing Octyl Glucoside (OG);

FIG. 15G shows single-channel water permeability of pR-PH channels;

FIG. 15H shows the reflectance (or rejection) of pR-PH channels in liposomes;

FIG. 16A shows a representative stopped flow light scattering curve for liposomes containing pS-PH channels and having different CLRs after exposure to 400mM sucrose in a hypertonic solution at 25 ℃;

FIG. 16B shows a representative stopped flow light scattering curve for liposomes containing pR-PH channels with different CLRs after exposure to 400mM sucrose in hypertonic solution at 25 ℃;

FIG. 16C shows the net water permeability of pR-PH in liposomes with different CLRs measured under hypertonic conditions at 25 ℃;

FIG. 17A is a schematic of a stopped flow light scattering test for swelling experiments;

FIG. 17B shows representative stopped flow light scattering curves for liposomes containing pS-PH channels (CLR 0 and 0.0025) after exposure to hypertonic solution (same buffer without 100mM NaCl) at 25 ℃;

FIG. 17C shows the net water permeability of pR-PH in liposomes (CLR 0.0025 and 0.005) in swelling and shrinking mode at 25 ℃;

FIG. 18A shows UV-vis spectra of pR-PH channel solutions (0. mu.M to 15. mu.M);

FIG. 18B shows a calibration curve of UV-vis absorbance versus pR-PH channel concentration;

FIG. 18C shows CD spectra of pR-PH channel solutions (0. mu.M to 15. mu.M);

FIG. 18D shows a calibration curve of CD absorbance versus pR-PH channel concentration;

FIG. 18E shows the size of the hypothetical liposomes and the surface areas of phosphatidylcholine/phosphatidylserine (PC/PS) lipids and pR-PH channels;

fig. 18F shows the insertion efficiency and number of pR-PH channels in liposomes at CLR 0.005;

figure 19 shows representative stopped flow light scattering curves for PR-PH channel containing liposomes (CLR ═ 0.005) exposed abruptly to hypertonic solutions of 200mM NaCl, 400mM glycine, 400mM valine, 400mM glucose and 400mM sucrose at 25 ℃ using different solutes as osmotic agents;

FIG. 20 shows a comparison of pR-PH channels with AQP and other artificial water channels.

Detailed Description

The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and changes may be made without departing from the scope of the present invention. The various embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more other embodiments to form new embodiments.

Features described in the context of one embodiment may apply correspondingly to the same or similar features in other embodiments. Features described in the context of one embodiment may be correspondingly applicable to other embodiments, even if not explicitly described in these other embodiments. Furthermore, additions and/or combinations and/or substitutions described for features in the context of one embodiment may be correspondingly applicable to the same or similar features in other embodiments.

The invention relates to a column [5]]Column of attachment peptides of derivatives of aromatic hydrocarbons [5]Aromatic hydrocarbon hydrolysates and a process for the synthesis of such hydrolysates. The term "hydrolysate" as used herein refers to a column [5] derived from an attached peptide]Hydrolyzed compounds of aromatic hydrocarbons. Advantageously, each hydrolysate derived from the present process provides 1.3X 10⁹Water permeability of water molecules/s, which is superior to column [5]]Conventional derivatives of aromatic hydrocarbons, and are of the same order of magnitude (i.e. 10)⁹) At least comparable to the water permeability of aquaporins. The hydrolysate also provides for substantial or even complete entrapment of salts and solutes. As used herein, the term "attached to a peptide" and grammatical variations thereof means a column [5]]The aromatic hydrocarbon is covalently bonded to the peptide via an amide bond. The term "amide" refers to a chemical group having the form-NHC (═ O) -.

The column [5] arene to which the peptide is attached can be used as a template to form a tubular structure of water channels by functionalization, for example to form hydroxyl or carboxyl groups on the attached peptide. The term "hydroxy" as used herein refers to an-OH functionality. The term "carboxyl" as used herein refers to the-COOH functional group. As disclosed herein, this column [5] arene-based water channel demonstrates superior water conductivity with desirable salt rejection over conventional column [5] arenes with water conductivity but without salt rejection. The phrase "water-conducting ability" as used herein refers to the ability to allow water molecules to pass through the channels defined by the tubular structure of the column [5] arene hydrolysis product to which the peptide is attached.

The hydrolysis products of the pillared [5] arenes attached to peptides disclosed herein have planar chirality due to the rotation of benzene units around the methylene bridge of the pillared [5] arenes. By binding bulky rigid peptides and their substituents to column [5] arenes, the two most stable isomers (abbreviated as pS and pR) (fig. 2A) can be formed, and since the energy of these two isomers is the lowest, it is preferred to form these two isomers by the present method. Indeed, the planar chirality of the hydrolysates disclosed herein is a factor affecting the water conducting properties of the water channels. The present synthesis of column [5] arene hydrolysis products of attached peptides disclosed herein separate the (pS) -and (pR) -isomers of column [5] arene, resulting in improved water conductivity and having desirable salt rejection, as these isomers of column [5] arene are specifically synthesized.

In the present invention, the phrase "liposome membrane" broadly refers to a membrane that contains lipids and can be bound to a hydrolysate. The lipid may be in the form of a liposome. The liposome membrane may be in the form of a membrane formed from lipids and bound to the hydrolysate. The liposome membrane may be in the form of a membrane comprising one or more liposomes associated with the hydrolysate. The liposome membrane may alternatively be liposomes associated with a hydrolysate. Thus, the phrase "liposomal membrane" may refer to any of the forms of membranes described above, and may be used interchangeably with the phrase "lipid membrane" in that the membrane comprises lipids. The phrases "liposome membrane" and "lipid membrane" may be referred to as "water membrane" because liposome membrane may be used to conduct water, e.g., to improve water flux in filtration.

In the present invention, the term "substantially" does not exclude "completely", e.g., a composition "substantially free" of Y may be completely free of Y. The term "substantially" may be omitted from the definition of the present invention, if necessary.

In the context of various embodiments, the articles "a," "an," and "the" used in reference to a feature or element include reference to one or more features or elements.

In the context of various embodiments, the term "about" or "approximately" as applied to a numerical value encompasses both the precise value and a reasonable variance.

As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Unless otherwise specified, the terms "include" and "comprise," as well as grammatical variants thereof, are intended to mean "open" or "inclusive" language such that it includes the recited elements but also allows for inclusion of additional, unrecited elements.

The details of the method of synthesizing the hydrolysate, the hydrolysate and the use thereof, and the method of determining the insertion efficiency of the hydrolysate in the liposome membrane and the respective embodiments of the present invention are described below.

In the present invention, a method for synthesizing a column [5] arene hydrolysate of attached peptide is provided. The method comprises the following steps: formation of a column [5] arene substituted with a carboxyl group from a dialkoxybenzene; mixing a column [5] arene substituted with a carboxyl group with a tripeptide organic solvent to form a column [5] arene attached to the peptide, wherein the tripeptide has a terminal-NH 2 group and a terminal ester; and hydrolyzing the peptide-attached column [5] arene in the presence of a base to obtain a peptide-attached column [5] arene hydrolysate.

In the present method, forming the carboxyl group-substituted column [5] arene may include mixing a dialkoxybenzene with paraformaldehyde in the presence of an organic acid catalyst to form the dialkoxy column [5] arene. The term "dialkoxybenzene" as used herein refers to a benzene having two alkoxy groups disposed opposite each other, wherein each alkoxy group has the form of an-O-alkyl group, and the "O" atom is directly attached to the benzene via a covalent bond. An example of such a dialkoxybenzene may be 1, 4-dimethoxybenzene. Thus, the term "dialkoxy column [5] arene" refers to a column [5] arene having two alkoxy groups disposed opposite each other, wherein the alkoxy groups have been defined above.

The term "alkyl" as used herein as a group or part of a group refers to a straight or branched chain aliphatic hydrocarbon group including, but not limited to: c₁-C₁₀Alkyl radical, C₁-C₉Alkyl radical, C₁-C₈Alkyl radical, C₁-C₇Alkyl radical, C₁-C₆Alkyl radical, C₁-C₅Alkyl radical, C₁-C₄Alkyl radical, C₁-C₃Alkyl and C₁-C₂An alkyl group. Suitable straight and branched chains C₁-C₆Examples of alkyl substituents include: methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, hexylAnd the like.

By placing the alkoxy groups opposite each other on the benzene ring, the hydrolysis products of the pillared [5] arenes attached to the peptide are configured to define a channel that does not impede the passage of water molecules, since the position of the alkoxy groups forms the site to which the peptide can be attached. In various embodiments, the dialkoxybenzene may include 1, 4-dimethoxybenzene. In embodiments where 1, 4-dimethoxybenzene is used, the dialkoxy [5] arene may comprise a 1, 4-dimethoxycolumn [5] arene.

In various embodiments, the organic acid catalyst may include boron trifluoride diethyl etherate, p-toluenesulfonic acid, or trifluoroacetic acid. As an example of an acid catalyst, trifluoroacetic acid may provide a higher yield of dialkoxy column [5] arene, which in turn provides a higher yield of hydrolysis products.

In the present method, forming the carboxyl group-substituted column [5] arene may include mixing a dialkoxy column [5] arene with boron tribromide to form the column [5] arene. As an example, boron tribromide can be used to completely remove the alkoxy groups to give higher yields of column [5] arene substituted with carboxyl groups. Reaction with boron tribromide converts the alkoxy groups on the dialkoxy column [5] arene to hydroxyl groups. Thus, the column [5] arene of the present invention may have two hydroxyl groups placed opposite each other on the benzene, and the advantages of this positioning have been described above. In various embodiments, the column [5] arene may be represented by the formula:

in various embodiments, a column substituted with a carboxyl group is formed [5]]The aromatic hydrocarbon may comprise subjecting the column [5] to a catalyst]Aromatic hydrocarbons are mixed with alkyl acetates to form carbonyl group substituted columns [5]An aromatic hydrocarbon. Alkyl acetates are low cost and easy to use in the present process. Formation of carbonyl group-substituted columns [5]]The aromatic hydrocarbon advantageously allows conversion into a carboxyl group, which can then be reacted with the "-NH" of the peptide₂"groups react to attach the peptide to the column [5]]On benzene of aromatic hydrocarbons.

The term "alkyl acetates"alkyl ester" means alkyl-C (═ O) O^-Acetate ion in its form. In various embodiments, the alkyl acetate may include butyl bromoacetate, ethyl bromoacetate, methyl bromoacetate, or propyl bromoacetate. Alkyl acetate and column [5]]The hydroxyl groups of the aromatic hydrocarbon react to form carbonyl groups.

In the context of the present invention, a carbonyl group means a group of the form "-R_mC(＝O)R_n- "wherein R is_mAnd R_nRepresents a general organic substituent including hydrogen. In various embodiments, a carbonyl group substituted column [5]]The aromatic hydrocarbon may comprise ethoxycarbonylmethoxy-substituted column [5]]An aromatic hydrocarbon.

In various embodiments, the catalyst may comprise potassium iodide or sodium iodide. The use of these iodides or other suitable iodides may help to increase the yield of carbonyl group substituted column [5] arene.

In the method, forming the carboxyl group-substituted column [5] arene may include mixing the carbonyl group-substituted column [5] arene with an alkaline solution to form the carboxyl group-substituted column [5] arene. As described above, when the column [5] arene becomes substituted with a carboxyl group, the carboxyl group allows the peptide to attach thereto. According to various embodiments, the alkaline solution may include cesium hydroxide, lithium hydroxide, potassium hydroxide, or sodium hydroxide.

In various embodiments, the carboxy group-substituted column [5] arene may be represented by the formula:

once the column substituted with carboxyl groups [5] was obtained]Aromatic hydrocarbons which can be mixed with peptides to form columns for attaching peptides [5]]An aromatic hydrocarbon. The peptide may be a tripeptide. The term "tripeptide" refers to a peptide having three amino acids connected by an amide bond, the peptide having a terminal-NH₂And a terminal ester. This tripeptide allows compatible incorporation of the resulting water channel into the liposome membrane. The term "terminal" as used herein means-NH₂The group and the ester are located at the end of the carbon chain of the peptide. This includes-NH₂The radicals and esters being situated three relative to one anotherThe position of the end of the carbon chain of the peptide. The term "ester", as used herein, whether as a group or as part of a group, such as in an "ester linkage", refers to a compound having-C (═ O) O-.

Mixing the carboxyl group-substituted column [5] arene with the tripeptide may include mixing the carboxyl group-substituted column [5] arene with the tripeptide at a temperature of 45 ℃ to 70 ℃ for 12 hours to 180 hours (hrs) under an inert atmosphere. The temperature can be 45 ℃ to 70 ℃, 50 ℃ to 70 ℃, 60 ℃ to 70 ℃, 45 ℃ to 60 ℃, 50 ℃ to 60 ℃, 45 ℃ to 50 ℃ and the like. The duration may be from 12hrs to 180hrs, from 50hrs to 180hrs, from 100hrs to 180hrs, from 150hrs to 180hrs, from 12hrs to 150hrs, from 12hrs to 100hrs, from 12hrs to 50hrs, from 50hrs to 150hrs, from 50hrs to 100hrs, from 100hrs to 150hrs, and so forth.

In various embodiments, the tripeptide may include NH₂-L-Phe-L-Phe-L-Phe-O-methyl, NH₂-L-Phe-L-Phe-L-Phe-O-ethyl, NH₂-L-Phe-L-Phe-L-Phe-O-propyl, NH₂-D-Phe-D-Phe-D-Phe-O-methyl, NH₂-D-Phe-D-Phe-D-Phe-O-ethyl, or NH₂-D-Phe-D-Phe-D-Phe-O-propyl. In various embodiments, the tripeptide may include NH₂-L-Phe-L-Phe-L-Phe-O-methyl. As an example, the peptide may make the water channel more stable, thereby forming more stable open pores when incorporated into liposomes.

In various embodiments, the organic solvent may include anhydrous dimethylformamide, 4-dimethylaminopyridine, and N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride. The advantage of these organic solvents is that, for example, dimethylformamide can be used to dissolve the various compounds disclosed herein, and 4-dimethylaminopyridine and N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride can result in higher yields of column [5] arene forming the attached peptide.

The inert atmosphere may include or may be nitrogen, argon, or any other non-reactive gas, such as a noble gas.

The method may further comprise: subjecting the column [5] arene with the attached peptide to column chromatography to produce a fraction comprising a diastereomeric mixture of the column [5] arene with the attached peptide, mixing the diastereomeric mixture with acetone, and then filtering the diastereomeric mixture to obtain a residue and a filtrate, wherein the residue comprises the diastereomer pR and the filtrate comprises the diastereomer pS. The fraction disclosed herein may be a first fraction that elutes from a chromatography column. Acetone as used herein is advantageous because acetone is a good solvent for separating two diastereomers based on their different solubilities. Diastereomer pS has good solubility in acetone, whereas diastereomer pR cannot dissolve in acetone.

The term "diastereomer" as used herein refers to stereoisomers of column [5] arene attached peptides including hydrolysates that have different planar configurations due to one or more, but not all, stereocenters and are not mirror images of each other. The term "diastereomer pR" refers to a diastereomer of column [5] arene with an attached peptide in the pR configuration. This diastereomer is shown in figure 2A. The term "diastereomer pS" refers to a diastereomer of a column [5] arene having an attached peptide with the pS configuration. This diastereomer is shown in figure 2A.

The separated diastereomers of the column [5] arene attached peptide may be hydrolyzed to obtain a hydrolysate, and in various embodiments, hydrolyzing the column [5] arene attached peptide may include mixing the column [5] arene attached peptide with a base for 8 hours to 40 hours (hrs). The duration may alternatively be, for example, from 10hrs to 40hrs, from 20hrs to 40hrs, from 30hrs to 40hrs, from 8hrs to 30hrs, from 8hrs to 20hrs, from 8hrs to 10hrs, from 10hrs to 30hrs, from 10hrs to 20hrs, from 20hrs to 30hrs, and the like

In various embodiments, the base may include lithium hydroxide, potassium hydroxide, or sodium hydroxide. These hydroxides selectively hydrolyze the esters and avoid unnecessary hydrolysis of the amides to form the resulting hydrolysis products.

The present invention also provides a hydrolysis product of a peptide-attached column [5] arene configured as a water channel in a liposome membrane, wherein the hydrolysis product of the peptide-attached column [5] arene can be represented by the following formula:

wherein R represents a tripeptide having a terminal-NH-group and a terminal-COOH group, wherein the terminal-NH-group forms part of an amide bond, and wherein the terminal-COOH group extends away from the amide bond. As shown in the formula above, the terminal-NH-group of the tripeptide is attached to a-C (═ O) -group extending away from the benzene to form an amide bond. The hydrolysate is obtained or obtainable according to the process described in the various embodiments of the first aspect.

Embodiments and advantages described in the context of the present method are similarly valid for the hydrolysate described herein and vice versa. Since various embodiments and advantages have been described above, they will not be described in detail for the sake of brevity.

In various embodiments, the tripeptide may include-NH-L-Phe-L-Phe-L-Phe-COOH or-NH-D-Phe-D-Phe-D-Phe-COOH. In such embodiments where the tripeptides include-NH-L-Phe-L-Phe-L-Phe-COOH or-NH-D-Phe-D-Phe-D-Phe-COOH, the hydrolysate can have the pR configuration, the pS configuration, or a mixture thereof.

In the present invention, a method of synthesizing a liposome membrane comprising a hydrolysate of column [5] arene attached to a peptide is provided. Embodiments and advantages described in the context of the present method of synthesizing a hydrolysate are similarly valid for the method of synthesizing a liposomal membrane, and vice versa. Since various embodiments and advantages have been described above, they will not be described in detail for the sake of brevity.

The method can comprise the following steps: synthesizing a hydrolysate of the column [5] arene attached to the peptide according to the method described in the various embodiments of the first aspect, mixing the hydrolysate with a plurality of lipids to form a mixture, forming a liposome membrane from the mixture, contacting the liposome membrane with a buffer to form a suspension comprising the hydrolysate and the plurality of lipids, and extruding the suspension through a membrane to form the liposome membrane.

In the present method, mixing the hydrolysate with the plurality of lipids may comprise mixing the hydrolysate with the plurality of lipids in a molar ratio of greater than 0 and up to 0.02. Non-limiting examples of molar ratios can range from 0.025 to 0.02, 0.005 to 0.02, 0.01 to 0.02, and the like. For example, liposomal membranes in liposome form made from these molar ratios advantageously provide water permeability comparable to aquaporin without compromising salt and solute retention. The entrapment of salts and solutes may be a substantial entrapment, or even a complete entrapment. Substantial rejection includes at least 95%, 99% or even 100% rejection of salts and solutes. In certain embodiments, the molar ratio may be 0.02.

In the present method, mixing the hydrolysate with the plurality of lipids may comprise dissolving the hydrolysate in one or more organic solvents. The one or more organic solvents may include chloroform and/or methanol. Such organic solvents have a low boiling point, allowing easy removal by evaporation. In various embodiments, the chloroform and/or methanol may be present in a ratio of 0:1 to 20: a volume ratio of 1 is present. When a mixture of chloroform and methanol is used in such a ratio, all of the various compounds used herein can be easily dissolved. In certain embodiments, the chloroform and methanol may be present in a ratio of 1: a volume ratio of 1 is present.

For the synthesis of liposomal membranes, e.g., as liposomes, the various lipids may include phosphatidylcholine and/or phosphatidylserine. In various embodiments, the plurality of lipids can include phosphatidylcholine and/or phosphatidylserine. Other lipids may be used. In embodiments using phosphatidylcholine and/or phosphatidylserine, the ratio of phosphatidylserine to phosphatidylcholine can be in the range of 0:1 to 100: 1 is present. In certain embodiments, the phosphatidylserine and phosphatidylcholine can be present in a ratio of 1: 4 are present in a molar ratio. Advantageously, liposomes present in such a molar ratio tend to be more stable.

After mixing the hydrolysate with various lipids, a liposome membrane may be formed. Forming the liposome membrane can include drying the mixture to remove the one or more organic solvents. Drying may be performed by subjecting the liposome membrane to vacuum. The vacuum pressure may be, for example, 0.08 MPa.

The liposome membrane can then be mixed with a buffer to form a suspension. In suspension, liposomes can be formed as a hydrophobic membrane surrounding an aqueous core, where the membrane consists of a lipid bilayer. The buffer may include (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), sodium chloride, and sodium azide. The pH of the suspension may range from 4.5 to 9.0. In certain embodiments, the pH of the suspension may be 7. Any suitable reagent for preparing a buffer to form such a liposome suspension may be used. In various embodiments, the method may further comprise incubating the suspension at 0 ℃ to 50 ℃ for 10 hours to 18 hours. In certain embodiments, the method may further comprise incubating the suspension at 4 ℃ for 10 hours to 18 hours. The duration may also be 12 hours to 18 hours, 14 hours to 18 hours, 16 hours to 18 hours, 10 hours to 12 hours, 10 hours to 14 hours, 10 hours to 16 hours, 12 hours to 16 hours, 14 hours to 16 hours, 12 hours to 14 hours, and the like. These durations may result in higher binding efficiency of the hydrolysate in the liposome membrane (e.g. liposomes).

In the present method, the liposomal membrane may then be obtained by extruding the suspension through the membrane. In various embodiments, extruding the suspension may include extruding the suspension more than once. Extrusion of the suspension through a membrane can help to obtain unilamellar liposomes, and repeated extrusion can help to obtain monodisperse unilamellar liposomes with uniform diameters.

In the present invention, there is also provided a liposomal membrane comprising a plurality of lipids and at least one hydrolysis product of a peptide-attached column [5] arene, wherein the at least one hydrolysis product of a peptide-attached column [5] arene is represented by the formula:

wherein R represents a tripeptide having a terminal-NH-group and a terminal-COOH group, wherein the terminal-NH-group forms an amide bond with the carbonyl carbon, and wherein the terminal-COOH group extends away from the amide bond. The liposomal membrane is obtained or obtainable according to the methods described in the various embodiments of the methods of synthesizing liposomal membranes disclosed herein above.

Embodiments and advantages described in the context of the present method of synthesizing a hydrolysate, the present method of synthesizing a liposomal membrane, and the hydrolysate of the present invention are similarly effective for the liposomal membranes described herein, and vice versa. Since various embodiments and advantages have been described above, they will not be described in detail for the sake of brevity.

In the liposomal membrane of the invention, the hydrolysate and the plurality of lipids can be present in a molar ratio of greater than 0 and up to 0.02. The molar ratio can also range from 0.025 to 0.02, 0.005 to 0.02, 0.01 to 0.02, and the like.

In various embodiments, the tripeptide may include-NH-L-Phe-L-Phe-L-Phe-COOH or-NH-D-Phe-D-Phe-D-Phe-COOH.

In various embodiments, the plurality of lipids can include phosphatidylcholine and/or phosphatidylserine. As already described above, the phosphatidylcholine and/or phosphatidylserine may be present in a ratio of 0:1 to 100: 1 is present. As already described above, phosphatidylserine and phosphatidylcholine can be present in a ratio of 1: 4 are present in a molar ratio.

The liposome membrane may comprise or be a unilamellar liposome. The liposomes can have a diameter of 80nm to 250 nm. Liposomes can have a diameter within this range.

The present invention further provides a method for determining the insertion efficiency of a hydrolysate of column [5] arene attached to a peptide obtained according to the method described in the various embodiments of the first aspect. The method comprises the following steps: mixing the liposome membrane obtained according to each embodiment described in the method of synthesizing a liposome membrane with a buffer solution; subjecting the buffer solution to a UV-visible spectrum having a UV-visible absorption wavelength of 293nm to detect UV-visible absorbance from the pi-pi transition of benzene; and correlating the UV-visible absorbance to a UV-visible absorbance-concentration standard curve to determine the efficiency of intercalation.

The buffer solution may comprise octyl glucoside. Octyl glucoside may be used as a surfactant to readily dissolve the hydrolysate. The buffer solution may also comprise a salt, such as sodium chloride, and may be any suitable phosphate buffer solution.

In the present invention, there is further provided a method for determining the insertion efficiency of the peptide-attached column [5] arene hydrolysate in the liposome membrane, obtained according to the method described in the various embodiments of the first aspect. The method comprises the following steps: mixing the liposome membrane obtained according to the method according to each embodiment described in the method of synthesizing a liposome membrane with a buffer solution; subjecting the buffer solution to circular dichroism spectroscopy at a circular dichroism absorption wavelength of 306nm to detect circular dichroism absorbance from pi-pi transition of benzene; and correlating the circular dichroism absorbance with a circular dichroism absorbance-concentration standard curve to determine the intercalation efficiency.

The buffer solution may comprise octyl glucoside. The buffer solution may also comprise a salt, such as sodium chloride, and may be any suitable phosphate buffer solution.

In both methods of determining the insertion efficiency of the hydrolysate in the liposome membrane, a UV-visible absorbance-concentration standard curve and a circular dichroism absorbance-concentration standard curve may be prepared by subjecting solutions having different concentrations of lipids to a UV-visible spectrum having a UV-visible absorption wavelength of, for example, 280 nm. The concentration may be, for example, 0mM to 1.5 mM. The solution may be any suitable phosphate buffer including octyl glucoside and a salt (e.g., sodium chloride). The preparation of the calibration curve (i.e. the standard curve) may involve the same steps as the method of determining the efficiency of intercalation by UV-visible absorbance and circular dichroism absorbance. UV-visible absorbance measurements based on a wavelength of 280nm can be obtained for different concentrations and can be used to establish a standard curve for the correlation.

In summary, the present invention includes the synthesis and isolation of (pS) -columns [5] comprising peptides]Aromatic hydrocarbons and (pR) -columns [5]]Aromatic hydrocarbon method, wherein the abbreviations "pS" and "pR" refer to the planar chiral diastereomers having the pS configuration and pR configuration, respectively, shown in fig. 2A. The method can comprise the following steps: (a) formation of carboxylic acid group-substituted columns [5] in DMF]Aromatic hydrocarbons and-NH₂A solution of a blocked tripeptide; (b) introducing 4-dimethylaminopyridine and N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride into the solution(ii) a (c) Pillars substituted with carboxylic acid groups [5]Aromatic hydrocarbons with-NH₂Reaction of amine-terminated tripeptides sufficient to produce a column of attached peptide [5]The reaction time and reaction temperature of the aromatic hydrocarbon; (d) purification of the peptide-containing column by column chromatography [5]]Aromatic hydrocarbons, and separating the (pS) -isomer and the (pR) -isomer based on a difference in solubility of the (pS) -isomer and the (pR) -isomer in an organic solvent.

-NH₂The blocked tripeptide may comprise three L-phenylalanines. The reaction time may be in the range of 12hrs to 180 hrs. The reaction temperature may be in the range of 45 ℃ to 70 ℃; the organic solvent used to separate the (pS) -configuration diastereomer and the (pR) -configuration diastereomer may be acetone. The (pR) -configuration diastereomer may form an insoluble solid in acetone. The (pR) -configuration diastereomer may be a peptide-attached (pR) -column [5]]An aromatic hydrocarbon. The (pS) -configuration diastereomer can form a soluble solid in acetone. The (pS) -configuration diastereomer may be a peptide-attached (pS) -column [5]]An aromatic hydrocarbon. (pR) -column [5] for peptide attachment]Aromatic hydrocarbons are highly water permeable and available selective water channels in the liposome membrane.

The liposome membrane may include attached peptide (pR) -pillared [5] arenes that are incorporated into the membrane with the liposomes.

The water permeability of (pR) -pillared [5] arenes attached to peptides can be measured by stop-flow light scattering experiments. The method may further comprise determining single channel water permeability by circular dichroism and UV-visualization techniques. The invention also provides for calculating activation energy by measuring water permeability at different temperatures and estimating relative solute retention by using different solutes as osmotic agents. The different temperatures may range from 10 ℃ to 25 ℃. Solutes used as osmotic agents may include sodium chloride, glycine, glycerol, glucose, and/or sucrose.

The liposomal membrane used, for example in the form of liposomes, may comprise L- α -phosphatidylcholine and L- α -phosphatidylserine. The molar ratio of L- α -phosphatidylcholine to L- α -phosphatidylserine may be 4: 1.

the resulting water permeability values may be in the range of 45 μm/s to 1200 μm/s. The single channel water permeability may be 3.9 x 10^-14cm³S, corresponding to 1.3X 10⁹Water molecules/s. (pR) -column [5] for peptide attachment]The activation energy of the aromatic hydrocarbon channels may be 7.77. + -. 1.06 kcal/mol. (pR) -column [5] for peptide attachment]The relative rejection of aromatics to sodium chloride may be greater than 1.

While the above methods are illustrated and described as a series of steps or events, it will be appreciated that any ordering of such steps or events is not to be interpreted in a limiting sense. For example, some steps may occur in different orders and/or concurrently with other steps or events apart from those illustrated and/or described herein. In addition, not all illustrated steps may be required to implement one or more aspects or embodiments described herein. Moreover, one or more steps depicted herein may be performed in one or more separate acts and/or phases.

Examples

The present invention relates to an artificial water channel comprising pillar [5] arene with attached peptide and a manufacturing method thereof.

In particular, the artificial water channel may be composed of a pillar [5] arene attached to a peptide in the pR configuration. These artificial water channels were found to have excellent water-conducting activity. These artificial water channels also show substantial entrapment of salts and small solutes. The water permeability of these artificial water channels ranges from 58 μm/s to 783 μm/s based on the lipid-channel molar ratio and the temperature at which the permeability measurements were obtained.

The water permeability of the individual water channels was found to be 1.3X 10⁹Water molecules/s/channel, which is permeable to water with aquaporins (4X 10)⁹Individual water molecules/s/channel) are equivalent.

The artificial water channel comprises a pillar [5] arene attached to a peptide, wherein the pillar [5] arene attached to the peptide is in a pR configuration and the peptide has a levorotatory (L) configuration. In certain embodiments, the peptide can be L-Phe-L-Phe-L-Phe-COOH.

As for the method of manufacturing the artificial water channel, the method may include dissolving the carboxylic acid group-substituted column [5] arene in anhydrous Dimethylformamide (DMF) to form a solution. The peptide, 4-Dimethylaminopyridine (DMAP), and N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) can be added to the solution to form a mixture, and the mixture can be stirred at 45 ℃ to 70 ℃ for 12hrs to 180hrs under an inert environment. The mixture can then be poured into hydrochloric acid (1 wt% to 10 wt%) to obtain a precipitate. The precipitate may be subjected to column chromatography to yield a first fraction comprising pS-methylated column [5] arene and pR-methylated column [5] arene artificial water channels. The first fraction may be filtered and washed with acetone to yield an insoluble solid comprising pR-methylated column [5] arene artificial water channels and a filtrate comprising pS-methylated column [5] arene. Lithium hydroxide monohydrate may be added to a solution of the pR-methylated column [5] arene artificial water channel, stirred at room temperature for 8 to 40hrs, concentrated under reduced pressure and acidified with 1 to 5 wt% hydrochloric acid to form the pR-unmethylated column [5] arene artificial water channel.

The invention also relates to a water membrane incorporating an artificial water channel. The water membrane may include an artificial water channel, wherein the artificial water channel comprises pR-unmethylated column [5] arene. The water membrane may be a lipid membrane, wherein the lipid membrane is formed from lipids or may be in the form of a membrane comprising liposomes incorporating water channels.

The invention also relates to a method for determining the efficiency of insertion of a peptide-attached column [5] arene into a liposome membrane, such as a liposome. The method may comprise the steps of: the column [5] arene and liposome membranes to which the peptides are attached are dissolved in phosphate buffer solution of octyl glucoside and the presence of the pi-pi transition of benzene is detected using the UV-vis (ultraviolet visible) method with an absorbance signal of about 293nm or using circular dichroism with an absorbance signal of 306 nm.

Details of the artificial water channel and its synthesis, the liposome membrane, and the method of determining the insertion efficiency are further discussed by the following non-limiting examples.

Example 1: material

In N₂Under the atmosphere, with CaH₂Distillation of methylene Chloride (CH)₂Cl₂). Trifluoroacetic acid (TFA), 1, 4-dimethoxybenzene, paraformaldehyde, boron tribromide (BBr)₃) Ethyl bromoacetate, 4-Dimethylaminopyridine (DMAP), anhydrous DMF and 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (4- (2-hydroxyethyl) -1-piperizineethanehanesulfonic acid, Hepes) from Sigma Aldrich.

L- α -Phosphatidylcholine (egg, PC) and L- α -phosphatidylserine (pig brain, sodium salt, PS) were purchased from Avanta polar Lipids.

O- (benzotriazol-1-yl) -N, N, N ', N ' -tetramethyluronium tetrafluoroborate (TBTU) and N-ethyl-N ' - (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from GL Biochem, Inc. Tripeptides (NH)₂L-Phe-L-Phe-L-Phe-OMe) was also available from GL Biochem (Shanghai) Ltd.

Water (18 M.OMEGA.cm) purified by Milli-Q system was used.

Polyethersulfone (PES, E6020P) was purchased from BASF and used to support the manufacture of membranes.

The materials were used as supplied by the supplier.

Example 2: device

Recording on a 500MHz Bruker DRX NMR spectrometer, a Bruker Avance III 400(400MHz) (100MHz) spectrometer or a Bruker AV-300(300MHz) spectrometer¹H and¹³c NMR spectrum. Low resolution mass spectra (LR-MS) were obtained on a ThermoFinnigan LCQ sweet MS. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) spectra were obtained in a positive helix mode at an acceleration potential of 20kV on JMS-S3000SpiralTOF (JEOL Ltd., Japan). LRMS and MALDI-TOFMS are reported in units of charge-to-mass ratio (m/z). The UV-Vis spectra were measured on a Cary Varian 5000 UV-Vis spectrometer using a 1cm quartz cuvette. Circular Dichroism (CD) spectroscopy was performed at room temperature in a cuvette with a 1cm path length using a Jasco J-1500CD spectrometer.

Example 3: method for synthesizing column [5] arene attached with peptide

The synthesis of (pS) -pillared [5] arenes and (pR) -pillared [5] arenes for attachment of peptides is described below with reference to the accompanying drawings. Specifically, the synthesis of (pS) -column [5] arene (pS-PH) and (pR) -column [5] arene (pR-PH) involved six steps, and an example of this synthesis is shown in fig. 1.

The first step involves the synthesis of 1, 4-dimethoxycolumn [5] arene (DMP 5).

DMP5 was prepared by using trifluoroacetic acid as a catalyst. Trifluoroacetic acid (15mL) was added to a solution of 1, 4-dimethoxybenzene (4.15g, 30mmol) and freshly ground paraformaldehyde (0.9g, 30mmol) in 1, 2-dichloroethane (285mL) and the mixture was refluxed at 90 ℃ for 3 hrs. After cooling to room temperature, the mixture was poured into methanol (400mL), and the resulting precipitate was collected by filtration and dissolved in CHCl₃(70 mL). Acetone (70mL) was added to remove from CHCl₃A precipitate was obtained and the solid was washed with acetone to give DMP5(3.25g) as a white solid. The yield was 69%.¹H NMR(500MHz,CDCl₃) 6.90(s,10H),3.77(s,10H),3.75(s, 30H). Calculating C by LR-MS (EI)₄₅H₅₁O₁₀[M+H]⁺: 751.35. obtaining: 751.31.

the second step involves a column [5]]Synthesis of aromatic hydrocarbons (P5). In this step, boron tribromide (13.2mL, 140mmol) was slowly added to DMP5(3.0g, 4mmol) in anhydrous CH₂Cl₂(100mL) in solution. The resulting mixture was stirred at room temperature for 72 hrs. Water (100mL) was then added slowly at 0 deg.C, and the mixture was stirred at room temperature for an additional 36 hrs. The precipitate was filtered and washed with water to give a milky white solid (2.4 g). The yield was 98%.¹H NMR(300MHz,CD₃COCD₃) 7.98(s,10H),6.68(s,10H),3.60(s, 10H). Calculating C by LR-MS (EI)₃₅H₃₄NO₁₀[M+NH₄]⁺: 628.22. obtaining: 628.26.

the third step involves an ethoxycarbonylmethoxy substituted column [5]]Synthesis of aromatic hydrocarbons (EP 5). Will K₂CO₃(7.0g) was added to a solution of P5(1.4g, 2.3mmol) in acetonitrile (60 mL). The mixture was stirred at room temperature for 1hr, then KI (80mg) and ethyl bromoacetate (5mL, 45mmol) were added. The mixture was heated to reflux under nitrogen for 24hrs and then left to cool at room temperature. The mixture was filtered and washed with CHCl₃And (6) washing. The filtrate was concentrated in vacuo and the residue was dissolved in CHCl₃(15 mL). By slow diffusion of methanol into CHCl₃In solution, a white solid formed. Collecting the white solid by filtrationMethanol was washed and dried under vacuum to give EP5 as a white product (2.74 g). The yield was 81%. H NMR (300MHz, CDCl)₃):＝7.04(s,10H),4.54(q,J＝15Hz,20H),4.09(m,J＝6Hz,20H),3.85(s,10H),0.96(m,J＝6Hz,30H)。

The fourth step was a carboxylic acid group substituted column [5]]Synthesis of aromatic hydrocarbons (AP 5). In this step, 20% aqueous sodium hydroxide (60mL) was added to a solution of EP5(2.7g, 1.8mmol) in THF (100 mL). The solution was heated to reflux for 15 hrs. After cooling, the solution was concentrated, diluted with water (100mL) and acidified with HCl. The white precipitate was collected by filtration, washed with water, and dried under vacuum to give AP5(1.9g) as a white solid. The yield was 87%.¹H NMR(300MHz,CD₃SOCD₃) 7.03(s,10H),4.64(d, J18 Hz,10H),4.41(d, J15 Hz,10H),3.72(s, 10H). Calculating C by LR-MS (EI)₅₅H₄₉O₃₀[M-H]^-: 1189.23. obtaining: 1189.33.

in the next step, the fifth step, pS-PM and pR-PM are synthesized. First, AP5(0.1mmol to 2mmol, 1 equiv.) was dissolved in anhydrous DMF. Reacting NH₂L-Phe-L-Phe-L-Phe-OMe (1mmol to 60mmol, 10 equiv. to 30 equiv.), DMAP (1mmol to 300mmol, 10 equiv. to 150 equiv.), and EDC (1mmol to 90mmol, 10 equiv. to 45 equiv.) were added to a solution of AP5 in anhydrous DMF. The term "equivalent" in this step means the amount used relative to the amount of AP 5. The mixture was stirred at 45 ℃ to 70 ℃ for 12hrs to 180hrs under nitrogen atmosphere. After cooling, the solution was poured into aqueous HCl (1% to 10%). The precipitate was filtered off and washed with water to give the crude product. The crude product was subjected to column chromatography. The first fraction (mixture of pS-PM and pR-PM) was collected and then dissolved in acetone. After filtration and thorough washing with acetone, insoluble solids were collected to obtain pR-PM. The filtrate was evaporated to give pS-PM.

In certain examples of synthesis and isolation of pS-PM and pR-PM, NH is₂L-Phe-L-Phe-L-Phe-OMe (3.26g, 6.8mmol), DMAP (3.5g, 28mmol) and EDC (1.34g, 7mmol) were added to a solution of AP5(0.44g, 0.37mmol) in anhydrous DMF (45 mL). The mixture was stirred at 55 ℃ for 86hrs under nitrogen atmosphere. After cooling, the solution was poured into aqueous HCl (2%, 400mL). The precipitate was filtered off and washed with water to give the crude product. The crude product is subjected to column Chromatography (CH)₂Cl₂:CH₃OH 10: 1). The first fraction (mixture of pS-PM and pR-PM) was collected and concentrated to give a white solid (0.9 g). The solid was then dissolved in acetone (50 mL). After filtration and thorough washing with acetone, insoluble solids were collected to give pR-PM (0.3 g). The filtrate was evaporated to give pS-PM (0.58 g).

pS-PM：¹H NMR(300MHz,CD₃SOCD₃):＝8.47-8.44(br,20H),7.56(br,10H),7.15-6.96(m,150H),6.73(s,10H),4.74-4.66(m,20H).4.54-4.44(m,10H),4.35(d,J＝15Hz,10H),4.20(d,J＝15Hz,10H),3.50(s,40H),2.98-2.77(m,60H)。¹³C NMR(100MHz,CD₃SOCD₃) 171.99,171.39,171.18,167.89,148.96,137.68,137.58,137.30,129.59,129.41,128.70,128.38,127.00,126.69,114.98,54.15,54.04,53.62,52.23,38.55,38.24 and 37.21. MALD-TOF-MS: calculate C₃₃₅H₃₄₀N₃₀O₆₀Na[M+Na]⁺: 5768.4463. obtaining: 5768.5095.

pS-PM：¹H NMR(300MHz,CD₃SOCD₃):＝8.50-8.40(br,20H),7.89(br,10H),7.22-6.95(m,150H),6.60(s,10H),4.67(br,20H).4.49(br,10H),4.20-4.09(br,20H),3.52(s,40H),2.95-2.78(m,60H)。¹³C NMR(100MHz,CD₃SOCD₃) 171.98,171.39,171.19,167.93,148.99,137.65,137.57,137.28,129.59,129.41,128.71,128.39,127.01,126.70,126.62,115.02,67.70,54.15,54.05,53.64,52.24,38.50,38.21 and 37.25. MALD-TOF-MS: calculate C₃₃₅H₃₄₀N₃₀O₆₀Na[M+Na]⁺: 5768.4463. obtaining: 5768.5073.

for the sixth step, pS-PH and pR-PH were obtained by adding lithium hydroxide monohydrate (0.02mmol-8mmol, 20 equiv-80 equiv.) and water to a solution of pS-PM or pR-PM (0.001mmol-0.1mmol, 1 equiv.) in THF. In this step, the term "equivalent" means the amount used relative to pS-PM or pR-PM. The mixture was stirred at room temperature for 8 to 40 hrs. The solution was then concentrated under reduced pressure and poured into water. After acidification with aqueous HCl (1 wt% to 5 wt%), the precipitate formed was filtered off, washed with water and dried under vacuum to give the white product.

An example of synthesizing pS-PH based on the above procedure is described below.

Lithium hydroxide monohydrate (30mg, 0.70mmol) and water (2.5mL) were added to a solution of pS-PM (80mg, 0.014mmol) in THF (7.5 mL). The mixture was stirred at room temperature for 20 hrs. Then, the solution was concentrated under reduced pressure and poured into water (30 mL). After acidification with aqueous HCl (2%), the precipitate formed was filtered off, washed with water and dried under vacuum to give the white product pS-PH (72.6mg, yield: 93%).¹H NMR(300MHz,CD₃SOCD₃):＝8.43(br,10H),8.31(br,10H),7.54(br,10H),7.19-6.95(m,150H),6.71(s,10H),4.71(br,20H).4.48(br,10H),4.35(br,10H),4.16(br,10H),3.51(s,10H),3.00-2.80(m,60H)。¹³C NMR(100MHz,CD₃SOCD₃) 173.06,171.33,170.90,168.15,148.92,137.62,136.83,129.59,129.43,128.58,128.39,126.80,114.88,67.62,54.18,53.07,38.16,38.08 and 37.31. MALD-TOF-MS: calculate C₃₂₅H₃₂₀N₃₀O₆₀Na[M+Na]⁺: 5628.2898. obtaining: 5628.2896.

an example of the synthesis of pR-PH based on the above procedure is described below.

Lithium hydroxide monohydrate (30mg, 0.70mmol) and water (2.5mL) were added to a solution of pR-PM (80mg, 0.014mmol) in THF (7.5 mL). The mixture was stirred at room temperature for 20 hrs. Then, the solution was concentrated under reduced pressure and poured into water (30 mL). After acidification with aqueous HCl (2%), the precipitate formed was filtered off, washed with water and dried under vacuum to give the white product pR-PH (71.8mg, yield: 92%).¹H NMR(300MHz,CD₃SOCD₃):＝8.35(br,20H),7.88(br,10H),7.19-6.94(m,150H),6.59(s,10H),4.64(m,20H).4.45(br,10H),4.20-4.10(br,20H),3.01-2.76(m,60H)。¹³C NMR(100MHz,CD₃SOCD₃) 173.13,171.34,171.19,167.92,148.95,137.73,137.56,129.60,129.52,128.62,128.38,126.86,126.67,114.98,67.61,54.14,53.60,38.46,38.17 and 37.34. MALD-TOF-MS: calculate C₃₂₅H₃₂₀N₃₀O₆₀Na[M+Na]⁺：5628.2898. Obtaining: 5628.2880.

example 4: method for preparing liposome membrane

Liposomal membranes, e.g., in liposome form, can be prepared by membrane rehydration (filmrehydration) as described below.

pR-PH or pS-PH channels in chloroform/methanol mixture (v/v-1/1) were added to 4 μmol PC/pS mixture at 4/1 molar ratio. The solvent was slowly distilled off on a rotary evaporator and subsequently dried under high vacuum to remove residual solvent. The dried film was washed with a solution containing 10mM Hepes (pH 7), 100mM NaCl and 0.01% NaN₃1mL of buffer (1) was rehydrated. The suspension was further incubated for 14hrs at 4 ℃ with stirring, then extruded 21 times through a 0.2 μm track etched membrane (Whatman, UK). The size of the liposomes was measured to be 160. + -.15 nm using a Nano Zetasizer (NanoZS, Malvern Instruments Limited, UK).

Example 5: stopped flow light scattering test program

The stall measurement is performed with a stall device (SX-20, Applied Photophysics) at a given temperature. The liposome solution was rapidly mixed with a hypertonic osmotic agent (400mM sucrose) in the same buffer to induce liposome contraction due to the osmotic gradient. The change in light scattering at a wavelength of 500nm was recorded. The stopped flow data was fitted to a single exponential function to obtain the rate constant (k). The water permeability (P) of the liposomes was calculated using the following equation (1)_f)：

Where k is the rate constant, S_o/V_oIs the ratio of initial surface area to liposome volume, V_wIs the partial molar volume of water (18 cm)³mol^-1) And Δ_osmIs the osmotic pressure difference. All osmolalities were measured using a freeze point osmometer (Model 3250, advanced measurements, inc.).

Example 6A: efficiency of insertion of channels

Liposomal membranes (e.g., in the form of liposomes) with and without pR-pH channels containing 2mM PC/PS lipids at a molar ratio of 4/1 were first prepared in phosphate buffer (10mM sodium phosphate, pH 6.4, 100mM NaCl) using the thin film rehydration method described above. The formed liposomes were further extruded 21 times through a 0.2 μm track-etched membrane (Whatman, UK) to obtain monodisperse unilamellar liposomes. The water permeability of the liposomes was studied by stopped flow measurements. Liposomes were exposed to a hypertonic osmotic agent (400mM sucrose). The net water permeability of pR-PH in liposomes (CLR ═ 0.005) was calculated to be 255.9 ± 26.4 μm/s.

To derive the insertion efficiency of the channel: 500 μ L of pR-pH channel solution (0 μ M to 30 μ M) in phosphate buffer containing 10mM sodium phosphate (pH 6.4), 100mM NaCl, and 8% n-octyl- β -D-glucoside (OG) was mixed with 500 μ L of control liposomes. The resulting solution was scanned on a UV-Vis spectrophotometer or a CD spectrophotometer (final concentration of channels: 0. mu.M to 15. mu.M). It should be noted that the channel-specific UV-Vis absorbance at 293nm and CD absorbance at 306nm increased linearly with concentration, generating a standard curve (FIG. 18). For channel-containing liposomes for water transport studies, 500 μ L of liposomes were mixed with 500 μ L of the same phosphate buffer containing 8% OG. The insertion efficiency of the channels into the liposomes was estimated based on the UV-vis absorbance at 292nm or the CD absorbance at 306nm, which are characteristic of the channels.

Insertion efficiency of lipid: a PC/PS lipid solution (0mM to 1.5mM) with a molar ratio of 4/1 was prepared in phosphate buffer containing 10mM sodium phosphate (pH 6.4), 100mM NaCl, and 4% n-octyl- β -D-glucoside (OG). The solution was scanned on a UV-vis spectrophotometer. The UV-vis absorbance at 280nm of the lipid solution increases with concentration and is proportional to concentration, generating a calibration curve. When extruded liposomes (initially 1mM) were measured at this wavelength, the actual concentration estimated from the calibration curve was 0.96 mM. The insertion efficiency of lipids was 96%. These steps are used to generate a standard calibration curve.

Example 6B: calculation of Single channel Water Permeability

Single-channel permeability was calculated based on pR-PH channel and lipid insertion efficiency. When the channel-lipid molar ratio, i.e., CLR, was 0.005, the radius (r) of the liposome was 80 nm. Assuming a bilayer thickness of 5nm, the outer and inner surfaceThe sum of the areas is 4 pi x r²+4π×(r-5)²＝151110nm². The average cross-sectional area of the lipid was 0.7nm on average²(the cross-sectional areas of PC and PS are about 0.7nm²) And the average cross-sectional area of the pR-PH channel was estimated to be 4.5nm²(FIG. 18E). The initial molar channel/lipid ratio was 1/200. If 96% of the lipids and 48% of the pR-PH channel are retained in the purified membrane vesicles, the actual molar channel/lipid ratio is 1/400. The number of channels inserted was 523/bubble. If the total net permeability of the channels in the liposomes is 255.9 μm/s, the single channel permeability is 3.9X 10^-14cm³S and 1.3X 10⁹Water molecules/s.

Example 7: solute entrapment

If small solutes are not completely trapped by the channel as osmotic agents, the osmotic gradient and measured water flux decrease. The solute rejection of the channel can be estimated from the reflection coefficient (σ). Sodium chloride, glycine, glycerol, glucose and sucrose were used as osmotic agents (fig. 19). Sucrose was used as reference solute (σ)_Sucrose1) and all experiments were measured at 25 ℃. The reflection coefficient is calculated by the following equation:

wherein sigma_SoluteIs the reflection coefficient of the solute, J_SoluteAnd J_SucroseThe water flux measured when solute and sucrose were used as osmotic agents, respectively.

Example 8A: discussion of methods of making Water channels

The synthesis and characterization of (pS) -pillared [5] arenes and (pR) -pillared [5] arenes for attachment of peptides is discussed below with reference to FIGS. 1-19.

Column for attachment of peptide [5]]The detailed synthesis of aromatics is shown in figure 1. Peptides attached to the column by amide condensation reactions between carboxylic acids and amines [5]On aromatic hydrocarbons. Collecting the first fraction by silica gel column chromatography, and passing through¹H NMR spectrum was analyzed (fig. 2C).¹HNMR spectroscopy identified the presence of two diastereomers in the first fraction, which was then further purified and fractionatedIsolating to obtain a (pS) -column [5] containing the peptide]Aromatic hydrocarbons and (pR) -columns [5]]Aromatic hydrocarbons, referred to as pS-PM and pR-PM, respectively (FIG. 2B). pS-PM was found to be the major product in the process due to stereoselectivity. The chemical structures of pS-PM and pR-PM are respectively passed through¹H NMR、¹³C NMR and mass analysis (fig. 3 to 8).

Example 8B: discussion of characterization results

Circular Dichroism (CD) spectra and UV-vis spectra were further used to characterize the structure of pS-PM and pR-PM. Both pR-PM and pS-PM showed affinity to column [5]]Pi-pi of aromatic radical fragment^*The transition corresponds to a characteristic UV-vis absorption band at 293nm (FIG. 2E). In the CD spectrum (FIG. 2D), a positive Koton effect for pR-PM (Cotton effect) and a negative Koton effect for pS-PM were observed, which is comparable to the previously reported bars [5]]The Ketton effect of aromatic derivatives is identical. The keton effect refers to a characteristic change in optical rotatory dispersion and/or circular dichroism near an absorption band of a substance. The coriolis effect is said to be positive if the optical rotation first increases as the wavelength decreases; the coriolis effect is said to be negative if the optical rotation first decreases as the wavelength decreases. These characterization results indicated complete isolation and purification of the diastereomers pR-PM and pS-PM.

The methyl groups in pS-PM and pR-PM were then easily removed by lithium hydroxide hydrolysis to provide the corresponding compounds pS-PH and pR-PH with free carboxylic acids that are expected to facilitate water transport through hydrogen bonding interactions between the carboxylic acid and water (fig. 2B). As shown in FIG. 2D, UV-vis and CD spectra of pS-PH and pR-PH confirmed that the conditions used for hydrolysis did not result in any change in configuration. In addition, of pS-PH¹H NMR Spectroscopy (FIG. 9) with peptide-attached column [5]]Process for preparing aromatic hydrocarbons¹The H NMR spectrum was consistent.

Example 8C: discussion of Water Permeability results

To study the water-transport behavior of both diastereomers, pS-PH and pR-PH were incorporated into phosphatidylcholine/phosphatidylserine (PC/pS) liposomes by lipid membrane rehydration in Hepes buffer, and the water permeability of the liposomes was studied using stop-flow light scattering experiments. In the contraction experiment, liposomes were rapidly exposed to hypertonic solution to cause contraction of the liposomes, which resulted in an increase in the light scattering signal over time (fig. 15A). The initial rise of the light scattering curve was fitted to a single exponential equation and the resulting exponential coefficient (k) was used to calculate the water permeability.

To obtain a low lipid background permeability, a low temperature (10 ℃) was first used. As shown in fig. 15B, since liposomes with different channel-to-lipid mole ratios (CLR of 0.005 and 0.01) containing pS-PH had almost the same light scattering curves (curves are overlapping) as the original liposomes, it was difficult to distinguish the contribution of pS-PH channel to water transport, meaning that the water-channeling activity of pS-PH channel was extremely low. However, binding of pR-PH channels significantly increased the overall water permeability compared to lipid background permeability (fig. 15C). The calculated permeability of liposomes containing a channel-to-lipid ratio (CLR) of pR-PH channels of 0.005 was 157.11 + -19.33 μm/s, which is 3 times greater than the calculated permeability (46.83 + -4.16 μm/s) of the original liposomes (46.83 + -4.16 μm). When the CLR was increased from 0.0025 to 0.02, the water permeability reached 446.99 ± 46.61 μm/s, which was almost 9 times higher than that of the original liposomes. Fig. 15D shows a gradual increase in clear water permeability obtained by excluding lipid background permeability.

To again confirm the high hydraulic activity of pR-PH, the measurement temperature was raised to 25 ℃ (fig. 16A to 16C), resulting in higher lipid background permeability. As expected, higher water channeling activity of pR-PH channels was observed in FIG. 16B. At CLR 0.02, the net water permeability reached 782.57 ± 124.78 μm/s (fig. 16C), which is orders of magnitude higher than conventional artificial water channels. The remarkably improved water permeability of the liposomes containing pR-PH channels means excellent water-conducting activity of the pR-PH channels.

To explore the water-conducting stability of the pR-PH channels, the water permeability of pR-PH channels in the swelling experiments was also studied by exposing liposomes to hypotonic solutions (fig. 17A to 17C). As a result, the water purification permeability values in the swelling mode and the shrinking mode were calculated to be the same order of magnitude, as shown in fig. 17C, suggesting stability of water-conducting activity of pR-PH channels in the lipid bilayer membrane environment.

Example 8D: discussion of activation energy calculation

The activation energies associated with water transport are assessed by measuring water permeability at different temperatures, and these energies can be used to determine whether transmembrane vesicle transport is diffusion driven or channel mediated. The contraction experiments were performed at different temperatures on the original liposomes and liposomes containing the pR-PH channel (CLR of 0.01) to generate Arrhenius plots (Arrhenius spot; ln k vs. 1/T) (FIG. 15E). The activation energy of the original liposomes was calculated to be 11.64. + -. 0.68kcal/mol, which is close to the previously reported activation energy value of the lipid bilayer membrane (12.39. + -. 2.21 kcal/mol). High activation energy indicates that water transport across the lipid bilayer is diffusion driven. Following incorporation of the pR-PH channel into liposomes, the activation energy was reduced to 7.77. + -. 1.06kcal/mol, which is strong evidence that water is channel-mediated through the pR-PH channel. This value is comparable to the reported value for AQP0 mediated water-conducting activation energy (7.60 ± 1.70kcal/mol), of which AQP0 is an example of the AQP family.

Example 8E: discussion of Single channel Water Permeability results

pR-PH channel dissolved in phosphate buffer containing Octyl Glucoside (OG) showed a characteristic UV-vis absorbance signal at 293nm and a CD absorbance signal at 306nm (FIGS. 18A and 18C). Thus, UV-vis and CD techniques are used to determine the efficiency of pR-PH channel insertion into the liposomal membrane (e.g., liposomal membrane form), which can be used to calculate single-channel water permeability. As expected, UV-vis absorbance at 293nm and CD absorbance at 306nm increased linearly with increasing concentration of pR-PH channels (FIGS. 18B and 18D). The insertion efficiency of the channels into liposomes determined by these two techniques exhibited similar values (UV-vis 48.13 + -7.76%, CD 49.20 + -4.53%) when the CLR was 0.005, as shown in FIG. 18F. The single-channel water permeability was calculated to be about 1.3 × 10 based on the insertion efficiency of the channel and the lipid⁹Water molecules/s/channel (figure 15G), which is comparable to AQP1 single channel water permeability (4 × 10)⁹Water molecules/s/channel) equivalent (see also fig. 20).

Example 8F: discussion of solute entrapment results

Reflection coefficients have been used to estimate the relative rejection properties of the channels. The determination of the reflection coefficient is based on the use of different solutesAs a stop flow light scattering experiment for penetrants (fig. 19). Sucrose was chosen as the reference solute due to its relatively large molecular size. The reflection coefficient was greater than 1 for all solutes (fig. 15F), indicating almost complete rejection of these solutes. The retention properties were attributed to the narrow pores of the pR-PH channel. The diameter of the narrowest region is about

This is very close to the diameter of AQP1

(see also FIG. 20).

Example 8G: discussion of pR-PH channel in comparison with other Water channels

The excellent properties of the pR-PH channel allowed us to compare it with biological aquaporins and previously reported artificial aquaporins (fig. 20). As a benchmark, aquaporin Z (AqpZ) was reconstituted in buffer as a PC/PS liposome, the properties of AqpZ are also presented in FIG. 20. The pR-PH channel was found to have as high water permeability as AQP and at least an order of magnitude higher than other artificial water channels. Note that the water permeability of the column [5] arene with the added peptide in the shrinkage mode is two orders of magnitude lower than in the swelling experiment due to the increase in low conductance channels. Unlike the column [5] arene with the peptide attached, the pR-PH channel showed similar water permeability values in the swelling mode and the contraction mode, suggesting stability of water-transport activity of the pR-PH channel in the lipid bilayer membrane environment. Furthermore, another advantage of pR-PH channels compared to other artificial water channels based on column [5] arenes without salt rejection is the excellent rejection of NaCl and small solutes, making it a viable material for incorporation into membrane materials for water purification applications.

Example 9: summary of the invention

The present invention and the examples described herein relate to the synthesis and characterization of (pR) -pillared [5] arenes as attachment peptides for highly permeable and selective artificial water channels. The (pS) -and (pR) -isomers of column [5] arenes containing peptides have been synthesized and isolated.

One aspect of the present invention provides a method for synthesizing (pS) -pillared [5] arenes and (pR) -pillared [5] arenes for attachment of peptides. Another aspect provides (pR) -pillared [5] arenes for use as attachment peptides for highly permeable and selective water channels. (pR) -pillared [5] arenes attached to peptides can be incorporated into the liposome membrane. Water permeability and selectivity can be measured by stop-flow light scattering experiments. Another aspect provides a UV-vis method for determining the efficiency of insertion of a peptide-attached (pR) -column [5] arene into a liposomal membrane (e.g., in liposomal form). In yet another aspect, a Circular Dichroism (CD) method of determining the insertion efficiency of peptide-attached (pR) -column [5] arene in a liposome membrane (e.g., liposome) is provided.

While the invention has been particularly shown and described with reference to a particular embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is, therefore, indicated by the appended claims, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. A method of synthesizing a hydrolysate of column [5] arene attached to a peptide, the method comprising:

column [5] substituted with said carboxyl groups in an organic solvent]Arene and tripeptide were mixed to form columns of attached peptides [5]An arene, wherein the tripeptide has a terminal-NH₂Groups and terminal esters; and

hydrolyzing the peptide-attached column [5] arene in the presence of a base to obtain the hydrolysis product of the peptide-attached column [5] arene.

2. The method of claim 1, wherein forming the carboxyl group-substituted column [5] arene comprises mixing the dialkoxybenzene with paraformaldehyde in the presence of an organic acid catalyst to form a dialkoxy column [5] arene.

3. A method according to claim 1 or 2, wherein the dialkoxybenzene comprises 1, 4-dimethoxybenzene.

4. The process of claim 1 or 2, wherein the organic acid catalyst comprises boron trifluoride diethyl etherate, p-toluenesulfonic acid, or trifluoroacetic acid.

5. The process according to any one of claims 2 to 4, wherein the dialkoxypillary [5] arene comprises a 1, 4-dimethoxypillared [5] arene.

6. The method of claim 1 or 2, wherein the column [5] arene is represented by the formula:

7. the method of any one of claims 1 to 6, wherein forming the carboxyl group-substituted column [5] arene comprises mixing the column [5] arene with an alkyl acetate in the presence of a catalyst to form a carbonyl group-substituted column [5] arene.

8. The method of claim 7, wherein the catalyst comprises potassium iodide or sodium iodide.

9. The method of claim 7 or 8, wherein the alkyl acetate comprises butyl, ethyl, methyl or propyl bromoacetate.

10. The method of any one of claims 7 to 9, wherein the carbonyl group-substituted column [5] arene comprises an ethoxycarbonylmethoxy-substituted column [5] arene.

11. The method of any one of claims 7 to 10, wherein forming the carboxyl group-substituted column [5] arene comprises mixing the carbonyl group-substituted column [5] arene with a basic solution to form the carboxyl group-substituted column [5] arene.

12. The method of claim 11, wherein the alkaline solution comprises cesium hydroxide, lithium hydroxide, potassium hydroxide, or sodium hydroxide.

13. The method of claim 1 or 2, wherein the carboxyl group-substituted column [5] arene is represented by the formula:

14. the method of any one of claims 1 to 13, wherein mixing the carboxyl group-substituted column [5] arene with the tripeptide comprises mixing the carboxyl group-substituted column [5] arene with the tripeptide under an inert atmosphere at a temperature of 45 ℃ to 70 ℃ for 12 hours to 180 hours.

15. The method of any one of claims 1 to 14, wherein the organic solvent comprises anhydrous dimethylformamide, 4-dimethylaminopyridine, and N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride.

16. The method of any one of claims 1 to 15, wherein the tripeptide comprises NH₂-L-Phe-L-Phe-L-Phe-O-methyl, NH₂-L-Phe-L-Phe-L-Phe-O-ethyl, NH₂-L-Phe-L-Phe-L-Phe-O-propyl, NH₂-D-Phe-D-Phe-D-Phe-O-methyl, NH₂-D-Phe-D-Phe-D-Phe-O-ethyl, or NH₂-D-Phe-D-Phe-D-Phe-O-propyl.

17. The method of any of claims 1 to 16, further comprising: subjecting the column [5] arene with the attached peptide to column chromatography to produce a fraction comprising a diastereomeric mixture of the column [5] arene with the attached peptide, mixing the diastereomeric mixture with acetone, and then filtering the diastereomeric mixture to obtain a residue and a filtrate, wherein the residue comprises the diastereomer pR and the filtrate comprises the diastereomer pS.

18. The method of any one of claims 1 to 17, wherein subjecting the peptide-attached column [5] arene to hydrolysis comprises mixing the peptide-attached column [5] arene with a base for 8 hours to 40 hours.

19. The method of any one of claims 1 to 18, wherein the base comprises lithium hydroxide, potassium hydroxide, or sodium hydroxide.

20. A hydrolysis product of a peptide-attached column [5] arene configured as a water channel in a liposome membrane, wherein the hydrolysis product of the peptide-attached column [5] arene is represented by the formula:

21. The hydrolysate of claim 20, wherein the tripeptide comprises-NH-L-Phe-COOH or-NH-D-Phe-COOH.

22. A method of synthesizing a liposomal membrane comprising a hydrolysate of column [5] arene attached to a peptide, the method comprising:

synthesizing a hydrolysate of column [5] arene attached to the peptide according to the method of any one of claims 1 to 19;

mixing the hydrolysate with a plurality of lipids to form a mixture;

forming a liposome membrane from the mixture;

contacting the liposome membrane with a buffer to form a suspension comprising the hydrolysate and the plurality of lipids; and

extruding the suspension through a membrane to form the liposomal membrane.

23. The method of claim 22, wherein mixing the hydrolysate with the plurality of lipids comprises dissolving the hydrolysate in one or more organic solvents.

24. The method of claim 23, wherein the one or more organic solvents comprise chloroform and/or methanol.

25. The method of claim 24, wherein the chloroform and methanol are present in a ratio of 0:1 to 20: a volume ratio of 1 is present.

26. The method of any one of claims 22 to 25, wherein the plurality of lipids comprises phosphatidylcholine and/or phosphatidylserine.

27. The method of claim 26, wherein the phosphatidylcholine and/or phosphatidylserine is present in a ratio of 0:1 to 100: 1 is present.

28. The method of any one of claims 22 to 27, wherein mixing the hydrolysate with the plurality of lipids comprises mixing the hydrolysate with the plurality of lipids in a molar ratio of greater than 0 and at most 0.02.

29. The method of any one of claims 22 to 28, wherein the buffer comprises (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), sodium chloride, and sodium azide.

30. The method of any one of claims 22 to 29, further comprising incubating the suspension at a temperature of 0 ℃ to 50 ℃ for 10 hours to 18 hours.

31. The method of any one of claims 22 to 30, wherein extruding the suspension comprises extruding the suspension more than once.

32. A liposomal membrane comprising a plurality of lipids and at least one hydrolysis product of a peptide-attached column [5] arene, wherein the at least one hydrolysis product of a peptide-attached column [5] arene is represented by the formula:

wherein R represents a tripeptide having a terminal-NH-group and a terminal-COOH group, wherein the terminal-NH-group forms an amide bond with a carbonyl carbon, and wherein the terminal-COOH group extends away from the amide bond.

33. The liposomal membrane of claim 32, wherein the tripeptide comprises-NH-L-Phe-COOH or-NH-D-Phe-COOH.

34. The liposomal membrane according to claim 32 or 33, wherein the plurality of lipids comprises phosphatidylcholine and/or phosphatidylserine.

35. Liposomal membrane according to claim 34, characterised in that the phosphatidylcholine and/or phosphatidylserine is present in a ratio of 0:1 to 100: 1 is present.

36. The liposomal membrane according to any one of claims 32-35 wherein the hydrolysate is present in a molar ratio to the plurality of lipids that is greater than 0 and at most 0.02.

37. The liposomal membrane according to any one of claims 32-36, wherein the liposomal membrane comprises unilamellar liposomes.

38. The liposomal membrane according to claim 37, wherein the diameter of the liposomes is from 80nm to 250 nm.