CN111902430B - Column [5] arene for high permeability and selective artificial water channel attachment peptides, synthesis and characterization thereof - Google Patents
Column [5] arene for high permeability and selective artificial water channel attachment peptides, synthesis and characterization thereof Download PDFInfo
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- CN111902430B CN111902430B CN201980020906.0A CN201980020906A CN111902430B CN 111902430 B CN111902430 B CN 111902430B CN 201980020906 A CN201980020906 A CN 201980020906A CN 111902430 B CN111902430 B CN 111902430B
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/06—Organic material
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
- B01D69/14—Dynamic membranes
- B01D69/141—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes
- B01D69/142—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers"
- B01D69/144—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers" containing embedded or bound biomolecules
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/06—Organic material
- B01D71/58—Other polymers having nitrogen in the main chain, with or without oxygen or carbon only
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/06—Organic material
- B01D71/74—Natural macromolecular material or derivatives thereof
Landscapes
- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Dispersion Chemistry (AREA)
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Abstract
According to the present invention, there is provided a method for synthesizing and isolating (pS) -pillar [5] arene and (pR) -pillar [5] arene attached with peptides. The peptide-attached (pR) -pillar [5] arene can form a single molecular channel with a nanotube structure for water transport. Methods of synthesizing (pR) -column [5] arene for the attached peptide are discussed herein that develop high permeability and selective water channels, the method comprising incorporating the (pR) -column [5] arene for the attached peptide into a liposome membrane, measuring the water permeability of the (pR) -column [5] arene for the attached peptide by a stopped-flow light scattering experiment, 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 rejection by employing different solutes as osmotic agents.
Description
Cross Reference to Related Applications
The present application claims the priority benefit of singapore patent application 10201802298R filed on day 21, 3, 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 a hydrolysate of column [5] arene of an attached peptide. The invention also relates to such hydrolysates and their use.
Background
Aquaporin (AQP) is defined by the narrowest constrictionAn hourglass-like structure. This unique structure not only allows for rapid water transport (about 10 8 -10 9 Molecules/second/channel) and also prevents solute transport. This transport property motivates extensive research into the engineering of AQP into synthetic membranes for desalination and water purification applications. Thereafter, several classes of AQP-based biomimetic membranes have been designed and manufactured with excellent water flux and desalination properties. However, the challenges of AQP production costs, low AQP stability, and AQP film fabrication have hampered the large-scale application of AQP.
Thus, artificial water pathways compatible for incorporation into lipid membranes configured with a water permeable central aperture and an outer hydrophobic shell are considered alternatives to AQP. Several classes of artificial water channels have been designed and developed based on chemical synthesis and self-assembly, improving the water-conducting rate and selectivity of the artificial water channels. However, conventional water channels are not capable of achieving water permeability and/or selectivity of AQP.
Moreover, the exploration of the artificial water channel is still in the conception verification stage and is far away 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 pathway 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 a peptide-attached column [5] arene, the method comprising:
forming a carboxyl group substituted column [5] arene from the dialkoxybenzene;
column substituted with carboxyl groups [5]]Aromatic hydrocarbon and tripeptide are mixed in an organic solvent to form a column [5] of attached peptide]Aromatic hydrocarbons in which the tripeptide has a terminal-NH 2 Groups and terminal esters; and
The peptide-attached column [5] arene is hydrolyzed 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 liposome membrane comprising a hydrolysate of a column [5] arene attached to a peptide, the method comprising:
Synthesizing a hydrolysate of peptide-attached column [5] arene 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 liposome membrane.
In another aspect, a liposome membrane is provided comprising a plurality of lipids and at least one hydrolysate of peptide-attached column [5] arene, wherein the at least one hydrolysate of 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.
In another aspect, there is provided a method of determining the efficiency of insertion of a hydrolysate of peptide-attached column [5] arene according to the method described in the various embodiments of the first aspect, the method comprising:
mixing a 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 pi-pi transition of benzene; and
The UV-visible absorbance was correlated with a UV-visible absorbance-concentration standard curve to determine the insertion efficiency.
In another aspect, there is provided a method of determining the efficiency of insertion of a hydrolysate of peptide-attached column [5] arene according to the method described in the various embodiments of the first aspect, the method comprising:
mixing a liposome membrane obtained according to the method described herein with a buffer solution;
subjecting the buffer solution to circular dichroism chromatography with a circular dichroism absorption wavelength of 306nm to detect circular dichroism absorbance from pi-pi transitions of benzene; and
The circular dichroism absorbance is 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 invention will be described with reference to the following drawings, in which:
FIG. 1 shows the synthesis of peptide-attached (pS) -pillar [5] arene and peptide-attached (pR) -pillar [5] arene (pS-PH and pR-PH);
FIG. 2A shows the (pS) -and (pR) -isomers of planar chiral column [5] arene;
FIG. 2B shows the chemical structures of the (pS) -pillar [5] arene of the attachment peptide and the (pR) -pillar [5] arene of the attachment peptide (i.e., pS-PM and pR-PM, respectively) and their corresponding hydrolysates (pS-PH and pR-PH, respectively);
FIG. 2C shows DMSO-d at 298K 6 Wherein pS-PM, pR-PM and part of a mixture of pS-PM and pR-PM 1 H NMR 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 the UV-vis spectra of pS-PM, pR-PM, pS-PH and pR-PH in THF at 298K;
FIG. 3 shows the effect of DMSO-d 6 pS-PM in (B) 1 H NMR spectroscopy;
FIG. 4 shows the effect of DMSO-d 6 pS-PM in (B) 13 C NMR spectrum;
FIG. 5 shows MALDI-TOF (matrix assisted laser Desorption/ionization time of flight mass spectrometry) mass spectra of pS-PM;
FIG. 6 shows the effect of DMSO-d 6 Middle pR-PM 1 H NMR spectroscopy;
FIG. 7 shows the effect of DMSO-d 6 Middle pR-PM 13 C NMR spectrum;
FIG. 8 shows MALDI-TOF mass spectrometry of pR-PM;
FIG. 9 shows the effect of DMSO-d 6 pS-PH in 1 H NMR spectroscopy;
FIG. 10 shows the measurement of DMSO-d 6 pS-PH in 13 C NMR spectrum;
FIG. 11 shows MALDI-TOF mass spectrometry of pS-PM;
FIG. 12 shows the effect of DMSO-d 6 mesopR-PH 1 H NMR spectroscopy;
FIG. 13 shows the formation of a polymer in DMSO-d 6 mesopR-PH 13 C NMR spectrum;
FIG. 14 shows MALDI-TOF mass spectrometry of pR-PH;
FIG. 15A is a schematic of a stopped flow light scattering test (stop-flow light scattering test) for shrinkage experiments;
FIG. 15B shows the stop-flow light scattering curves of liposomes containing pS-PH channels and having different Channel-to-lipid molar ratios (Channel-to-lipid molar ratio, CLR) after exposure to a hypertonic solution of 400mM sucrose at 10 ℃;
FIG. 15C shows stopped-flow light scattering curves for liposomes containing pR-PH channels and having different channel-to-lipid molar ratios (CLR) after exposure to 400mM sucrose hypertonic solution at 10 ℃;
FIG. 15D shows the clean water permeabilities of pR-PH in liposomes with different channel-to-lipid molar ratios (CLR) measured at 10deg.C under hypertonic conditions;
FIG. 15E shows an Arrhenius relationship graph for calculating activation energy;
FIG. 15F is a schematic representation of solubilized pR-PH channel in a buffer containing Octyl Glucoside (OG);
FIG. 15G shows the single channel water permeability of pR-PH channels;
FIG. 15H shows reflectance (or retention) of pR-PH channels in liposomes;
FIG. 16A shows representative stop-flow light scattering curves for liposomes containing pS-PH channels and having different CLRs after exposure to a hypertonic solution of 400mM sucrose at 25 ℃;
FIG. 16B shows representative stop-flow light scattering curves for pR-PH channel containing liposomes with different CLRs after exposure to 400mM sucrose hypertonic solution at 25 ℃;
FIG. 16C shows the clean water permeabilities of pR-PH in liposomes with different CLRs measured at 25℃under hypertonic conditions;
FIG. 17A is a schematic of a stopped flow light scattering test for swelling experiments;
FIG. 17B shows representative stop-flow light scattering curves for pS-PH channel-containing liposomes (CLR 0 and 0.0025) after exposure to Gao Shenrong (same buffer without 100mM NaCl) at 25 ℃;
FIG. 17C shows the clean 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 putative liposomes and the surface areas of phosphatidylcholine/phosphatidylserine (PC/PS) lipids and pR-PH channels;
FIG. 18F shows the efficiency and number of pR-PH channels inserted into liposomes at CLR=0.005;
FIG. 19 shows representative stop-flow light scattering curves for PR-PH channel containing liposomes (CLR=0.005) using different solutes as osmotic agents, the liposomes being suddenly exposed to hypertonic solutions of 200mM NaCl, 400mM glycine, 400mM valine, 400mM glucose and 400mM sucrose at 25 ℃.
FIG. 20 shows a comparison of pR-PH channel 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 be correspondingly applicable 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 with respect to 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 for peptide adhesion of aromatic hydrocarbon derivative [5]]A hydrolysate of aromatic hydrocarbon and a method for synthesizing the hydrolysate. The term "hydrolysate" as used herein refers to a column derived from an attachment peptide [5]]A hydrolyzed compound of an aromatic hydrocarbon. Advantageously, each hydrolysate derived from the present process provides 1.3X10 9 The water permeability of water molecules/s is superior to that of 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 substantial or even complete entrapment of salts and solutes. As used herein, the term "peptide-attached" 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 peptide-attached column [5] arene can be used as a template to form the tubular structure of the water channel by functionalization, for example to form hydroxyl or carboxyl groups on the attached peptide. The term "hydroxy" as used herein refers to the-OH functionality. The term "carboxy" as used herein refers to the-COOH functional group. As disclosed herein, such column [5] arene-based water channels demonstrate superior water conductivity with the desired salt rejection over conventional column [5] arenes with water conductivity but without salt rejection. The phrase "water-conducting capacity" as used herein refers to the ability to allow water molecules to pass through the channel defined by the tubular structure of the hydrolysis product of the column [5] arene to which the peptide is attached.
The hydrolysis products of the peptide-attached column [5] aromatics disclosed herein have planar chirality due to the rotation of benzene units around the methylene bridge of the column [5] aromatics. By binding the bulky rigid peptide and its substituents to the column [5] arene, the two most stable isomers (abbreviated to pS and pR) can be formed (fig. 2A), 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 the hydrolysis products of column [5] arenes attached to peptides disclosed herein separates the (pS) -and (pR) -isomers of column [5] arenes, resulting in improved water conductivity and desired salt rejection, as these isomers of column [5] arenes are specifically synthesized.
In the present invention, the phrase "liposome membrane" refers broadly to a membrane that comprises 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 bound to the hydrolysate. The liposome membrane may alternatively be a liposome bound to the hydrolysate. Thus, the phrase "liposome membrane" may refer to any of the forms of membranes described above, and may be used interchangeably with the phrase "lipid membrane" because the membrane comprises lipids. The phrases "liposome membrane" and "lipid membrane" may be referred to as "water membrane" because liposome membranes can be used to conduct water, e.g., improve water flux in filtration.
In the present invention, the term "substantially" does not exclude "complete", e.g., a composition that is "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" as used with respect to a feature or element include references 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 an exact 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 indicated, the terms "comprise" and "comprising" and grammatical variants thereof are intended to mean "open" or "inclusive" language such that they include the recited elements but also allow for the inclusion of additional, unrecited elements.
Details of the methods of synthesizing a hydrolysate, the hydrolysate and uses thereof, and methods of determining the efficiency of insertion of the hydrolysate in a liposome membrane, and various embodiments of the invention are described below.
In the present invention, there is provided a method for synthesizing a hydrolysate of a peptide-attached column [5] arene. The method comprises the following steps: forming a carboxyl group substituted column [5] arene from the dialkoxybenzene; mixing a carboxy group substituted column [5] arene with a tripeptide organic solvent to form an attached peptide column [5] arene, 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 process, forming the carboxyl group substituted column [5] arene may include mixing dialkoxybenzene with paraformaldehyde in the presence of an organic acid catalyst to form the dialkoxycolumn [5] arene. The term "dialkoxybenzene" as used herein refers to benzene having two alkoxy groups placed 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 placed 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: c (C) 1 -C 10 Alkyl, C 1 -C 9 Alkyl, C 1 -C 8 Alkyl, C 1 -C 7 Alkyl, C 1 -C 6 Alkyl, C 1 -C 5 Alkyl, C 1 -C 4 Alkyl, C 1 -C 3 Alkyl and C 1 -C 2 An alkyl group. Suitable straight-chain and branched C 1 -C 6 Examples of alkyl substituents include: methyl, ethyl, n-propyl, 2-propyl, n-butylSec-butyl, tert-butyl, hexyl and the like.
By placing the alkoxy groups opposite each other on the benzene ring, the hydrolysis product of the peptide-attached column [5] arene is configured to define a channel that does not impede the passage of water molecules, as 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 column [5] arene may comprise 1, 4-dimethoxy column [5] arene.
In various embodiments, the organic acid catalyst may include boron trifluoride etherate, p-toluene sulfonic acid, or trifluoroacetic acid. As an example of an acid catalyst, trifluoroacetic acid may provide higher yields of dialkoxy column [5] arene, which in turn provides higher yields of hydrolysis products.
In the present process, 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 alkoxy groups to give higher yields of carboxyl group substituted column [5] arene. The reaction with boron tribromide converts the alkoxy groups on the dialkoxy column [5] arene into hydroxyl groups. Thus, the column [5] aromatics of the invention may have two hydroxyl groups placed opposite each other on benzene, and the advantages of such positioning have been described above. In various embodiments, the column [5] arene may be represented by the following formula:
in various embodiments, a carboxyl group substituted column [5] is formed]The aromatic hydrocarbon may comprise subjecting the column [5] to a reaction in the presence of a catalyst]Aromatic hydrocarbons are mixed with alkyl acetate to form carbonyl group substituted columns [5]]Aromatic hydrocarbons. Alkyl acetate is low cost and is readily available for use in the present process. Formation of carbonyl group substituted column [5] ]The aromatic hydrocarbon advantageously allows conversion to carboxyl groups, which can then be combined with the "-NH" of the peptide 2 "groups react to attach the peptide to the column [5]]Benzene of aromatic hydrocarbon.
The term "alkyl acetate" refers to alkyl-C (=o) O - Acetate ion in the 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, carbonyl groups refer to the form "-R m C(＝O)R n - "wherein R is m And R is n Represents a general organic substituent, including hydrogen. In various embodiments, carbonyl group substituted column [5]]Aromatic hydrocarbons may include ethoxycarbonylmethoxy substituted columns [5]]Aromatic hydrocarbons.
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] arenes.
In the present method, forming the carboxyl group-substituted column [5] arene may include mixing the carbonyl group-substituted column [5] arene with a basic 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 following formula:
once the carboxyl group substituted column [5] is obtained]Aromatic hydrocarbons, which can be mixed with peptides to form peptide-attached columns [5]]Aromatic hydrocarbons. The peptide may be a tripeptide. The term "tripeptide" refers to a peptide having three amino acids connected by amide linkages, the peptide having a terminal-NH 2 And terminal esters. Such tripeptides allow compatible incorporation of the resulting water channel into the liposome membrane. The term "terminal" as used herein means-NH- 2 Radicals and esters at carbon of peptidesThe end of the chain. This includes-NH 2 The group and the ester are located opposite each other at the end of the carbon chain of the tripeptide. The term "ester", as used herein, refers to a compound having-C (=o) O-, whether as a group or as part of a group, for example in an "ester linkage".
Mixing the carboxyl group-substituted column [5] arene with the tripeptide may include 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 (hrs). The temperature may be 45 to 70 ℃, 50 to 70 ℃, 60 to 70 ℃, 45 to 60 ℃, 50 to 60 ℃, 45 to 50 ℃, etc. The duration may be 12hrs to 180hrs, 50hrs to 180hrs, 100hrs to 180hrs, 150hrs to 180hrs, 12hrs to 150hrs, 12hrs to 100hrs, 12hrs to 50hrs, 50hrs to 150hrs, 50hrs to 100hrs, 100hrs to 150hrs, etc.
In various embodiments, the tripeptide may include NH 2 -L-Phe-L-Phe-L-Phe-O-methyl, NH 2 -L-Phe-L-Phe-L-Phe-O-ethyl, NH 2 -L-Phe-L-Phe-L-Phe-O-propyl, NH 2 -D-Phe-D-Phe-D-Phe-O-methyl, NH 2 -D-Phe-D-Phe-D-Phe-O-ethyl, or NH 2 -D-Phe-D-Phe-D-Phe-O-propyl. In various embodiments, the tripeptide may include NH 2 -L-Phe-L-Phe-L-Phe-O-methyl. By way of 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 attachment 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: column chromatography of peptide-attached column [5] arene is performed to produce a fraction comprising a diastereomeric mixture of peptide-attached column [5] arene, the diastereomeric mixture is mixed with acetone, and the diastereomeric mixture is then filtered to yield a residue and a filtrate, wherein the residue comprises diastereomer pR, and the filtrate comprises diastereomer pS. The fraction disclosed herein may be a first fraction eluted from the chromatographic column. Acetone as used herein is advantageous because acetone is a good solvent for separating two diastereomers based on their different solubilities. Diastereoisomer pS has good solubility in acetone, whereas diastereoisomer pR is not soluble in acetone.
The term "diastereoisomer" as used herein refers to stereoisomers of the pillar [5] arene, including the peptide of attachment of the hydrolysis product, which stereoisomers have different planar configurations due to one or more but not all of the stereocenters and are not mirror images of each other. The term "diastereomer pR" refers to a diastereomer of a column [5] arene having an attachment peptide of pR configuration. This diastereomer is shown in figure 2A. The term "diastereomer pS" refers to the diastereomer of a column [5] arene having the attachment peptide of pS configuration. This diastereomer is shown in figure 2A.
The separated diastereomers of the peptide-attached column [5] arene may be hydrolyzed to give a hydrolysate, and in various embodiments, hydrolyzing the peptide-attached column [5] arene may include mixing the peptide-attached column [5] arene with a base for 8 hours to 40 hours (hrs). The duration may alternatively be, for example, 10 to 40hrs, 20 to 40hrs, 30 to 40hrs, 8 to 30hrs, 8 to 20hrs, 8 to 10hrs, 10 to 30hrs, 10 to 20hrs, 20 to 30hrs, etc
In various embodiments, the base may include lithium hydroxide, potassium hydroxide, or sodium hydroxide. These hydroxides selectively hydrolyze esters and avoid unnecessary hydrolysis of 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 a 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 above formula, the terminal-NH-group of the tripeptide is attached to a-C (=o) -group extending away from benzene to form an amide bond. The hydrolysate is obtained or obtainable according to the method described in the various embodiments of the first aspect.
The embodiments and advantages described in the context of the present process are similarly valid for the hydrolysates described herein and vice versa. Since various embodiments and advantages have been described above, a detailed description is omitted for brevity.
In the context of the various embodiments of the present invention, tripeptides may include-NH-L-Phe-L-Phe-L-Phe-COOH or-NH-D-Phe-D-Phe-D-Phe-COOH. In such embodiments where the tripeptide includes-NH-L-Phe-L-Phe-L-Phe-COOH or-NH-D-Phe-D-Phe-D-Phe-COOH, the hydrolysis product may 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 a column [5] arene to which a peptide is attached is provided. The embodiments and advantages described in the context of the present method of synthesizing a hydrolysate are similarly valid for the method of synthesizing a liposome membrane and vice versa. Since various embodiments and advantages have been described above, a detailed description is omitted for brevity.
The method may include: synthesizing a hydrolysate of peptide-attached column [5] arene 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 a liposome membrane.
In the present method, mixing the hydrolysate with the plurality of lipids may include mixing the hydrolysate with the plurality of lipids in a molar ratio 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 so forth. For example, liposome membranes in the form of liposomes made from these molar ratios advantageously provide comparable water permeability to aquaporins without compromising on salt and solute entrapment. The entrapment of salts and solutes can be substantial entrapment, or even complete entrapment. Substantial entrapment includes entrapment of at least 95%, 99% or even 100% 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 include 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 for easy removal by evaporation. In various embodiments, chloroform and/or methanol may be used at 0:1 to 20:1 by volume. 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, chloroform and methanol can be used in the amount of 1:1 by volume.
For the synthesis of liposome membranes, for example as liposomes, the various lipids may include phosphatidylcholine and/or phosphatidylserine. In various embodiments, the plurality of lipids may include phosphatidylcholine and/or phosphatidylserine. Other lipids may be used. In embodiments using phosphatidylcholine and/or phosphatidylserine, the phosphatidylserine and/or phosphatidylcholine may be present at 0:1 to 100:1 is present in a molar ratio. In certain embodiments, phosphatidylserine and phosphatidylcholine can be present in an amount of 1:4 is present in a molar ratio. Advantageously, liposomes present in such molar ratios tend to be more stable.
After mixing the hydrolysate with the plurality of 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, 0.08MPa.
The liposome membrane can then be mixed with a buffer to form a suspension. In suspension, the liposomes can be formed as a hydrophobic membrane surrounding an aqueous core, where the membrane consists of a lipid bilayer. Buffers may include (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic 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, etc. These durations may result in higher binding efficiency of the hydrolysate in the liposome membrane (e.g. liposome).
In the present method, the liposome 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 diameter.
In the present invention, there is also provided a liposome membrane comprising a plurality of lipids and at least one hydrolysate of peptide-attached column [5] arene, wherein the at least one hydrolysate of 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. The liposome membrane is obtained or obtainable according to the methods described in the various embodiments of the methods of synthesizing a liposome membrane disclosed herein above.
The embodiments and advantages described in the context of the present method of synthesizing a hydrolysate, the present method of synthesizing a liposome membrane, and the hydrolysate of the present invention are similarly valid for the liposome membrane described herein, and vice versa. Since various embodiments and advantages have been described above, a detailed description is omitted for brevity.
In the liposome membrane of the present invention, the hydrolysate and the plurality of lipids may be present in a molar ratio of greater than 0 and up to 0.02. The molar ratio may also range from 0.025 to 0.02, 0.005 to 0.02, 0.01 to 0.02, etc.
In the context of the various embodiments of the present invention, tripeptides 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 may include phosphatidylcholine and/or phosphatidylserine. As already described above, phosphatidylcholine and/or phosphatidylserine may be present at 0:1 to 100:1 is present in a molar ratio. As already described above, phosphatidylserine and phosphatidylcholine can be present in a ratio of 1:4 is present in a molar ratio.
The liposome membrane may comprise or may be a unilamellar liposome. The liposomes can have a diameter of 80nm to 250 nm. Liposomes can have diameters within this range.
The invention further provides a method of determining the efficiency of insertion of the hydrolysis product of peptide-attached column [5] arene according to the methods described in the various embodiments of the first aspect. The method comprises the following steps: mixing a liposome membrane obtained according to the various embodiments 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 pi-pi transition of benzene; and correlating the UV-visible absorbance with a UV-visible absorbance-concentration standard curve to determine the insertion efficiency.
The buffer solution may comprise octyl glucoside. Octyl glucoside can be used as a surfactant to easily 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 of determining the efficiency of insertion of the hydrolysis product of the peptide-attached column [5] arene in a liposome membrane obtained according to the methods described in the various embodiments of the first aspect. The method comprises the following steps: mixing a liposome membrane obtained according to the method according to the various embodiments described in the method of synthesizing a liposome membrane with a buffer solution; subjecting the buffer solution to circular dichroism chromatography at a circular dichroism absorption wavelength of 306nm to detect circular dichroism absorbance from pi-pi transitions 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 efficiency of intercalation of the hydrolysis product in the liposome membrane, the UV-visible absorbance-concentration standard curve and the circular dichroism absorbance-concentration standard curve can be prepared by subjecting solutions having different concentrations of lipids to UV-visible spectrum having a UV-visible absorption wavelength of, for example, 280 nm. The concentration may be, for example, 0mM to 1.5mM. The solution may be any suitable phosphate buffer including octyl glucoside and salts (e.g., sodium chloride). The preparation of the calibration curve (i.e., standard curve) may involve the same steps as the method of determining the insertion efficiency by UV-visible absorbance and circular dichroism absorbance. UV-visible absorbance measurements based on a 280nm wavelength can be obtained for different concentrations and can be used to establish a correlation standard curve.
In summary, the invention includes the synthesis and isolation of (pS) -columns comprising peptides [5 ]]Aromatic hydrocarbons and (pR) -columns [5]The method of arenes, wherein the abbreviations "pS" and "pR" refer to the planar chiral diastereomers with pS and pR configurations, respectively, shown in fig. 2A. The method may include: (a) Formation of a column substituted with carboxylic acid groups in DMF [5 ]]Aromatic hydrocarbons and-NH 2 Blocked tripeptidesA solution; (b) Introducing 4-dimethylaminopyridine and N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride to the solution; (c) Column substituted with carboxylic acid group [5 ]]Aromatic hydrocarbons and-NH 2 Amine-terminated tripeptide reactions are sufficient to produce a column of attached peptide [5]Reaction time and reaction temperature of 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 the solubility difference of the (pS) -isomer and the (pR) -isomer in the organic solvent.
-NH 2 The capped tripeptide may include three L-phenylalanine. 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) -configurational diastereomers and the (pR) -configurational diastereomers may be acetone. The (pR) -configurational diastereomer may form an insoluble solid in acetone. The (pR) -configurational diastereoisomer may be a peptide-attached (pR) -column [5 ] ]Aromatic hydrocarbons. The (pS) -configurational diastereomer may form a soluble solid in acetone. The (pS) -configurational diastereoisomer may be a peptide-attached (pS) -column [5]]Aromatic hydrocarbons. Peptide-attached (pR) -column [5]Aromatic hydrocarbons are highly water permeable and available selective water channels in liposome membranes.
The liposome membrane may include (pR) -pillar [5] arene that is incorporated into the membrane along with the liposome.
The water permeability of the peptide-attached (pR) -column [5] arene can be measured by a stopped-flow light scattering experiment. The method may further comprise determining single channel water permeability by circular dichroism and UV-visible techniques. The invention also provides for calculating the activation energy by measuring water permeability at different temperatures and estimating the relative solute rejection 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 liposome membrane used, for example in the form of a liposome, may include L- α -phosphatidylcholine and L- α -phosphatidylserine. The molar ratio of L- α -phosphatidylcholine to L- α -phosphatidylserine may be 4:1.
obtaining The water permeability value of (2) may be in the range 45 μm/s to 1200 μm/s. The single channel water permeability can be 3.9X10 -14 cm 3 S is equivalent to 1.3X10 9 Water molecules/s. Peptide-attached (pR) -column [5]The activation energy of the aromatic hydrocarbon channels may be 7.77.+ -. 1.06kcal/mol. Peptide-attached (pR) -column [5]The relative entrapment of the aromatic hydrocarbon to sodium chloride may be greater than 1.
While the above method is illustrated and described as a series of steps or events, it will be appreciated that any ordering of such steps or events should not 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 pathway for column [5] aromatics comprising an attachment peptide and a method for manufacturing the same.
In particular, the artificial water pathway may consist of a pR-configured peptide-attached column [5] arene. These artificial water channels were found to have excellent water conducting activity. These artificial water pathways also exhibit substantial entrapment of salts and small solutes. The water permeabilities of these artificial water channels ranged from 58 μm/s to 783 μm/s based on the lipid-channel molar ratio and the temperature at which the permeabilities measurements were obtained.
The water permeability of the individual water channels was found to be 1.3X10 9 Individual water molecules/s/channel, which is compatible with the water permeability of aquaporins (4X 10 9 Individual water molecules/s/channel).
The artificial water pathway includes a peptide-attached column [5] arene, wherein the peptide-attached column [5] arene is in pR configuration and the peptide has a L configuration. In certain embodiments, the peptide may 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. Peptides, 4-Dimethylaminopyridine (DMAP) and N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) may be added to the solution to form a mixture, and the mixture may be stirred under an inert environment at 45 to 70 ℃ for 12 to 180hrs. The mixture may then be poured into hydrochloric acid (1 wt% to 10 wt%) to obtain a precipitate. The precipitate may be subjected to column chromatography to obtain 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 obtain insoluble solids comprising pR-methylation column [5] arene artificial water channels and a filtrate comprising pS-methylation column [5] arene. Lithium hydroxide monohydrate can be added to a solution of pR-methylated column [5] arene artificial water channel, stirred at room temperature for 8hrs to 40hrs, concentrated under reduced pressure and acidified with 1wt% to 5wt% hydrochloric acid to form 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 comprise 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 of lipids or may be in the form of a membrane comprising liposomes bound with water channels.
The invention also relates to a method for determining the efficiency of insertion of peptide-attached column [5] arene into a liposome membrane, such as a liposome. The method may comprise the steps of: the peptide-attached column [5] arene and liposome membrane were dissolved in a phosphate buffer solution of octyl glucoside, and the presence of pi-pi transition of benzene was detected with an absorbance signal of about 293nm using the UV-vis (ultraviolet visible) method, or with an absorbance signal of 306nm using circular dichroism.
Details of the artificial water channel and its synthesis, liposome membranes, and methods of determining the efficiency of insertion are further discussed by the following non-limiting examples.
Example 1: material
At N 2 Under the atmosphere, with CaH 2 Distillation of dichloromethane (CH) 2 Cl 2 ). Trifluoroacetic acid (TFA), 1, 4-dimethoxybenzene, paraformaldehyde, boron tribromide (BBr) 3 )、Ethyl bromoacetate, 4-Dimethylaminopyridine (DMAP), anhydrous DMF, and 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (4- (2-hydroxyyethyl) -1-piperazineethanesulfonic acid, hepes) were purchased from Sigma Aldrich.
L-alpha-phosphatidylcholine (egg, PC) and L-alpha-phosphatidylserine (pig brain, sodium salt, PS) were purchased from Avanti 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 (Shanghai) Inc. Tripeptide (NH) 2 -L-Phe-L-Phe-L-Phe-OMe) is also available from GL Biochem (Shanghai) Inc.
Water purified by Milli-Q system (18 mΩ cm) was used.
Polyethersulfone (PES, E6020P) was purchased from BASF for support membrane manufacture.
The materials were used in the manner provided by the suppliers.
Example 2: apparatus and method for controlling the operation of a device
Recording on a 500MHz Bruker DRX NMR spectrometer, a Bruker Avance III 400 (400 MHz) (100 MHz) spectrometer or a Bruker AV-300 (300 MHz) 1 H and 13 c NMR spectrum. Low resolution mass spectra (Low resolution mass spectra, LR-MS) were obtained at ThermoFinnigan LCQ Fleet MS. Matrix assisted laser Desorption/ionization time of flight mass spectrometry (Matrix assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF-MS) spectra were obtained in a positive helical mode on JMS 3000SpiralTOF (JEOL Co., ltd., japan) at an acceleration potential of 20 kV. LRMS and MALDI-TOFMS are reported in terms of charge-to-mass ratio (m/z). The UV-visible spectrum was measured on a Cary Varian 5000 UV-visible spectrometer using a 1cm quartz cuvette. Circular dichroism (Circular dichroism, CD) spectroscopy was performed at room temperature in a cuvette of 1cm path length using a Jasco J-1500CD spectrometer.
Example 3: method for synthesizing column [5] arene of adhesion peptide
The synthesis of (pS) -pillar [5] arene and (pR) -pillar [5] arene attached with peptides is described below with reference to the accompanying drawings. Specifically, the synthesis of (pS) -pillar [5] arene (pS-PH) and (pR) -pillar [5] arene (pR-PH) involves six steps, and an example of this synthesis is shown in FIG. 1.
The first step involves the synthesis of 1, 4-dimethoxy column [5] arene (DMP 5).
DMP5 was prepared by using trifluoroacetic acid as a catalyst. Trifluoroacetic acid (15 mL) was added to a solution of 1, 4-dimethoxybenzene (4.15 g,30 mmol) and freshly ground paraformaldehyde (0.9 g,30 mmol) in 1, 2-dichloroethane (285 mL) and the mixture was refluxed for 3hrs at 90 ℃. After cooling to room temperature, the mixture was poured into methanol (400 mL) and the resulting precipitate was collected by filtration and dissolved in CHCl 3 (70 mL). Acetone (70 mL) was added to remove the catalyst from the CHCl 3 The precipitate was washed with acetone to give DMP5 (3.25 g) as a white solid. The yield was 69%. 1 H NMR(500MHz,CDCl 3 ) δ=6.90 (s, 10H), 3.77 (s, 10H), 3.75 (s, 30H). LR-MS (EI) calculate C 45 H 51 O 10 [M+H] + :751.35. the method comprises the following steps: 751.31.
the second step involves a column [5]]Synthesis of aromatic hydrocarbons (P5). In this step, boron tribromide (13.2 mL,140 mmol) was slowly added to the anhydrous CH of DMP5 (3.0 g,4 mmol) 2 Cl 2 (100 mL) in solution. The resulting mixture was stirred at room temperature for 72hrs. Water (100 mL) was then slowly added at 0deg.C and the mixture was stirred at room temperature for an additional 36hrs. The precipitate was filtered and washed with water to give a milky white solid (2.4 g). The yield was 98%. 1 H NMR(300MHz,CD 3 COCD 3 ) δ=7.98 (s, 10H), 6.68 (s, 10H), 3.60 (s, 10H). LR-MS (EI) calculate C 35 H 34 NO 10 [M+NH 4 ] + :628.22. the method comprises the following steps: 628.26.
the third step involves ethoxycarbonylmethoxy substituted column [5 ]]Synthesis of aromatic hydrocarbons (EP 5). Will K 2 CO 3 (7.0 g) was added to a solution of P5 (1.4 g,2.3 mmol) in acetonitrile (60 mL). The mixture was stirred at room temperature for 1hr, followed by addition of KI (80 mg) and ethyl bromoacetate (5 mL,45 mmol). The mixture was heated to reflux under nitrogen for 24hrs, then left to cool at room temperature. The mixture was filtered and taken up in CHCl 3 And (5) washing. The filtrate was concentrated in vacuo and the residue was dissolved in CHCl 3 (15mL). By slow diffusion of methanol into CHCl 3 In solution, a white solid formed. The white solid was collected by filtration, washed with methanol, and dried under vacuum to give EP5 as a white product (2.74 g). The yield was 81%. H NMR (300 MHz, CDCl) 3 ):δ＝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 is a column substituted with carboxylic acid groups [5 ]]Synthesis of aromatic hydrocarbons (AP 5). In this step, 20% aqueous sodium hydroxide (60 mL) was added to a solution of EP5 (2.7 g,1.8 mmol) in THF (100 mL). The solution was heated to reflux for 15hrs. After cooling, the solution was concentrated, diluted with water (100 mL) and acidified with HCl. The white precipitate was collected by filtration, washed with water, and dried under vacuum to give white solid AP5 (1.9 g). The yield was 87%. 1 H NMR(300MHz,CD 3 SOCD 3 ) δ=7.03 (s, 10H), 4.64 (d, j=18 hz, 10H), 4.41 (d, j=15 hz, 10H), 3.72 (s, 10H). LR-MS (EI) calculate C 55 H 49 O 30 [M-H] - :1189.23. the method comprises the following steps: 1189.33.
in the next step, the fifth step, pS-PM and pR-PM are synthesized. First, AP5 (0.1 mmol to 2mmol,1 eq.) was dissolved in anhydrous DMF. NH is added to 2 -L-Phe-L-Phe-L-Phe-OMe (1 mmol to 60mmol,10 equivalents to 30 equivalents), DMAP (1 mmol to 300mmol,10 equivalents to 150 equivalents) and EDC (1 mmol to 90mmol,10 equivalents to 45 equivalents) were added to an anhydrous DMF solution of AP 5. The term "equivalent" in this step means the amount used relative to AP 5. The mixture was stirred under nitrogen at 45 to 70 ℃ for 12 to 180hrs. 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. A first fraction (mixture of pS-PM and pR-PM) was collected and then dissolved in acetone. After filtration and extensive washing with acetone, insoluble solids were collected to give pR-PM. The filtrate was evaporated to give pS-PM.
In certain examples of synthesis and isolation of pS-PM and pR-PM, NH is used 2 -L-Phe-L-Phe-L-Phe-OMe (3.26 g,6.8 mmol), DMAP (3.5 g,28 mmol) and EDC (1.34 g,7 mmol) were added to AP5 (0.44 g,0.37 mmol) in anhydrous form DMF (45 mL) solution. The mixture was stirred at 55℃for 86hrs under nitrogen atmosphere. After cooling, the solution was poured into aqueous HCl (2%, 400 mL). The precipitate was filtered off and washed with water to give the crude product. The crude product was subjected to column chromatography (CH 2 Cl 2 :CH 3 Oh=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 extensive 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： 1 H NMR(300MHz,CD 3 SOCD 3 ):δ＝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)。 13 C NMR(100MHz,CD 3 SOCD 3 ) Delta= 171.99,171.39,171.18,167.89,148.96,137.68,137.58,137.30,129.59,129.41,128.70,128.38,127.00,126.69,114.98,54.15,54.04,53.62,52.23,38.55,38.24,37.21.MALD-TOF-MS: calculate C 335 H 340 N 30 O 60 Na[M+Na] + :5768.4463. the method comprises the following steps: 5768.5095.
pS-PM： 1 H NMR(300MHz,CD 3 SOCD 3 ):δ＝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)。 13 C NMR(100MHz,CD 3 SOCD 3 ) Delta= 171.98,171.39,171.19,167.93,148.99,137.65,137.57,137.28,129.59,129.41,128.71,128.39,127.01,126.70,126.62,115.02,67.70,54.15,54.05,53.64,52.24,38.50,38.21,37.25.MALD-TOF-MS: calculate C 335 H 340 N 30 O 60 Na[M+Na] + :5768.4463. the method comprises the following steps: 5768.5073.
for the sixth step, pS-PH and pR-PH were obtained by adding lithium hydroxide monohydrate (0.02 mmol-8mmol,20 equivalents-80 equivalents) and water to a solution of pS-PM or pR-PM (0.001 mmol-0.1mmol,1 equivalent) 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 8hrs to 40hrs. The solution was then concentrated under reduced pressure and poured into water. After acidification with aqueous HCl (1 to 5 wt%) the precipitate formed is filtered off, washed with water and dried under vacuum to give a white product.
An example of the synthesis of pS-PH based on the above procedure is described below.
Lithium hydroxide monohydrate (30 mg,0.70 mmol) and water (2.5 mL) were added to a solution of pS-PM (80 mg,0.014 mmol) in THF (7.5 mL). The mixture was stirred at room temperature for 20hrs. 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 (72.6 mg, yield: 93%). 1 H NMR(300MHz,CD 3 SOCD 3 ):δ＝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)。 13 C NMR(100MHz,CD 3 SOCD 3 ) Delta= 173.06,171.33,170.90,168.15,148.92,137.62,136.83,129.59,129.43,128.58,128.39,126.80,114.88,67.62,54.18,53.07,38.16,38.08,37.31.MALD-TOF-MS: calculate C 325 H 320 N 30 O 60 Na[M+Na] + :5628.2898. the method comprises the following steps: 5628.2896.
an example of pR-PH synthesis based on the above procedure is described below.
Lithium hydroxide monohydrate (30 mg,0.70 mmol) and water (2.5 mL) were added to a solution of pR-PM (80 mg,0.014 mmol) in THF (7.5 mL). The mixture was stirred at room temperature for 20hrs. 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 pR-PH as a white product (71.8 mg, yield: 92%). 1 H NMR(300MHz,CD 3 SOCD 3 ):δ＝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)。 13 C NMR(100MHz,CD 3 SOCD 3 ):δ＝173.13,171.34,171.19,167.92,148.95,137.73,137.56,129.60,129.52,128.62,128.38,126.86,126.67,114.98,67.61,54.14,53.60,38.46,38.17,37.34.MALD-TOF-MS: calculate C 325 H 320 N 30 O 60 Na[M+Na] + :5628.2898. the method comprises the following steps: 5628.2880.
example 4: method for preparing liposome membrane
Liposome membranes, e.g., in liposome form, can be prepared by membrane rehydration (film rehydration) as described below.
pR-PH or pS-PH channels in chloroform/methanol mixtures (v/v=1/1) were added to 4. Mu. Mol of PC/PS mixture at a molar ratio of 4/1. The solvent was distilled slowly on a rotary evaporator and then dried under high vacuum to remove the residual solvent. The dried film was treated with a solution containing 10mM Hepes (pH=7), 100mM NaCl and 0.01% NaN 3 Is rehydrated with 1mL of buffer. The suspension was further incubated for 14hrs at 4℃with stirring and then extruded 21 times through a 0.2 μm track etched film (Whatman, UK). The liposomes were measured to be 160.+ -.15 nm in size using a Nano Zetasizer (NanoZS, malvern Instruments Limited, UK).
Example 5: stop-flow light scattering test program
The stop flow measurement is performed with a stop flow device (SX-20,Applied Photophysics) at a given temperature. The liposome solution was rapidly mixed with a hypertonic osmotic agent (400 mM sucrose) in the same buffer to induce shrinkage of the liposomes due to osmotic gradients. The change in light scattering at a wavelength of 500nm was recorded. The stop stream data is fitted to a single exponential function to obtain a rate constant (k). The water permeability (P) of the liposome was calculated using the following equation (1) f )：
Where k is the rate constant, S o /V o Is the ratio of initial surface area to liposome volume, V w Is the partial molar volume of water (18 cm 3 mol -1 ) And delta osm Is the osmotic pressure difference. All osmotic pressures were measured using a freeze point osmometer (Model 3250,Advanced Instruments.Inc.).
Example 6A: efficiency of insertion of channels
Liposome membranes (e.g., in liposome form) with and without pR-pH channels containing 2mM PC/PS lipids in a 4/1 molar ratio were first prepared in phosphate buffer (10 mM sodium phosphate, ph=6.4, 100mM NaCl) using the above-described membrane rehydration method. The liposomes formed were further extruded 21 times (Whatman, UK) through a 0.2 μm track etched membrane to give monodisperse unilamellar liposomes. The water permeability of liposomes was studied by stop flow measurement. The liposomes were exposed to a hypertonic osmotic agent (400 mM sucrose). The clean 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: mu.L of pR-PH channel solution (0. Mu.M to 30. Mu.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. Mu.L of control liposomes. The resulting solution was scanned on a UV-Vis spectrophotometer or 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 the channel-containing liposomes used in the water transport study, 500 μl of liposomes were mixed with 500 μl of the same phosphate buffer containing 8% og. The efficiency of channel insertion into liposomes was estimated based on the channel-specific UV-vis absorbance at 292nm or CD absorbance at 306 nm.
Efficiency of lipid insertion: PC/PS lipid solution (0 mM to 1.5 mM) was prepared at a molar ratio of 4/1 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 the concentration, generating a calibration curve. When the extruded liposomes (initially 1 mM) were measured at this wavelength, the actual concentration estimated from the calibration curve was 0.96mM. The lipid intercalation efficiency was 96%. These steps are used to generate a standard calibration curve.
Example 6B: calculation of Single channel Water Permeability
pR-PH channel and lipid based insertionAnd (5) calculating the single-channel permeability by efficiency. When the channel-lipid molar ratio, CLR, was 0.005, the radius (r) of the liposome was 80nm. Assuming a bilayer thickness of 5nm, the sum of the outer surface area and the inner surface area is 4pi×r 2 +4π×(r-5) 2 ＝151110nm 2 . The average cross-sectional area of the lipids was 0.7nm on average 2 (the cross-sectional areas of PC and PS are about 0.7nm 2 ) And the average cross-sectional area of pR-PH channel was estimated to be 4.5nm 2 (FIG. 18E). The initial channel/lipid molar ratio was 1/200. If 96% of the lipids and 48% of pR-PH channels were retained in the purified vesicles, the actual channel/lipid molar ratio was 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 3.9X10 -14 cm 3 S and 1.3X10 9 Water molecules/s.
Example 7: solute rejection
If small solutes are not completely trapped by the channel as osmotic agents, the osmotic gradient and measured water flux decreases. 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) Sucrose =1), and all experiments were measured at 25 ℃. The reflection coefficient is calculated as follows:
wherein sigma Solute (solute) Is the reflection coefficient of solute, J Solute (solute) And J Sucrose The water flux was 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 the (pS) -pillar [5] arene and (pR) -pillar [5] arene of the attachment peptide is discussed below with reference to fig. 1-19.
Peptide-attached column [5]]The detailed synthesis of aromatic hydrocarbons is shown in figure 1. The peptide is attached to the column by an amide condensation reaction between carboxylic acid and amine [5]]On aromatic hydrocarbons. The first fraction was collected by silica gel column chromatography,and pass through 1 The H NMR spectrum was analyzed (fig. 2C). 1 H NMR spectra identified the presence of both diastereomers in the first fraction, and then further purified and separated the two diastereomers to give a peptide-containing (pS) -column [5] ]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 present process due to stereoselectivity. The chemical structures of pS-PM and pR-PM are determined by 1 H NMR、 13 C NMR and mass analysis (FIGS. 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. pR-PM and pS-PM both showed a pattern similar to that of column [5 ]]Pi-pi of aryl fragments of aromatic hydrocarbons * The transition corresponds to a characteristic UV-vis absorption band at 293nm (FIG. 2E). In the CD spectrum (FIG. 2D), the positive Keyton effect of pR-PM (Cotton effect) and the negative Keyton effect of pS-PM were observed, which were comparable to the previously reported column [5 ]]The Keyton effect of the aromatic hydrocarbon derivatives is consistent. The Keyton effect refers to a characteristic change in optical dispersion and/or circular dichroism near the absorption band of a substance. If the optical rotation increases first as the wavelength decreases, the Keyton effect is referred to as positive; the koton effect is referred to as negative if the optical rotation decreases first with decreasing wavelength. These characterization results indicate complete isolation and purification of diastereomers pR-PM and pS-PM.
The methyl groups in pS-PM and pR-PM are then readily removed by lithium hydroxide hydrolysis to provide the corresponding compounds pS-PH and pR-PH with the free carboxylic acid that is expected to promote water transport through hydrogen bonding interactions between carboxylic acid and water (FIG. 2B). As shown in FIG. 2D, UV-vis and CD spectra of pS-PH and pR-PH confirm that the conditions used for hydrolysis did not result in any change in configuration. In addition, pS-PH 1 H NMR spectrum (FIG. 9) and column for attaching peptide [5 ]]Aromatic hydrocarbons 1 The H NMR spectra were consistent.
Example 8C: discussion of Water permeability results
To study the water-guiding behavior of both diastereomers, pS-PH and pR-PH were incorporated into phosphatidylcholine/phosphatidylserine (PC/pS) liposomes by lipid film rehydration in Hepes buffer and the water permeability of the liposomes was studied using a stop-stream light scattering experiment. In the shrinkage experiments, liposomes were rapidly exposed to hypertonic solutions to cause shrinkage of the liposomes, which resulted in an increase in light scattering signal over time (fig. 15A). The initial rise of the light scattering curve is fitted to a single exponential equation and the resulting exponential coefficient (k) is used to calculate the water permeability.
To obtain low lipid background permeability, first a low temperature (10 ℃) was used. As shown in fig. 15B, since liposomes containing pS-PH with different channel-lipid molar ratios (CLR of 0.005 and 0.01) have almost the same light scattering curves (the curves overlap) as the original liposomes, it is difficult to distinguish the contribution of pS-PH channels to water transport, meaning that the water-conducting activity of pS-PH channels is very low. However, the binding of pR-PH channels significantly increased the total water permeability compared to the lipid background permeability (fig. 15C). The calculated permeability of liposomes containing pR-PH channels with channel-to-lipid ratio (CLR) of 0.005 was 157.11 + -19.33 μm/s, exceeding the calculated permeability (46.83+ -4.16 μm/s) of the original liposomes (46.83+ -4.16 μm). When CLR is increased from 0.0025 to 0.02, the water permeability reaches 446.99 ±46.61 μm/s, which is almost 9 times higher than that of the original liposome. Fig. 15D shows that the permeability of the purified water obtained by removing the lipid background permeability gradually increases.
To again confirm the high water-conducting activity of pR-PH, the measurement temperature was raised to 25 ℃ (fig. 16A to 16C), resulting in a higher lipid background permeability. As expected, higher water-conducting activity of pR-PH channel was observed in FIG. 16B. At clr=0.02, the clean water permeability reached 782.57 ± 124.78 μm/s (fig. 16C), which is several orders of magnitude higher than that of the conventional artificial water channel. The significantly improved water permeability of liposomes containing pR-PH channels suggests excellent water-conducting activity of pR-PH channels.
To explore the water-guiding stability of pR-PH channels, the water permeability of pR-PH channels in swelling experiments was also studied by exposing liposomes to hypotonic solutions (FIGS. 17A-17C). As a result, the clear water permeability values in the swelling mode and the shrinkage mode were calculated to be of the same order, suggesting stability of the water-conducting activity of pR-PH channel in the lipid bilayer membrane environment as shown in FIG. 17C.
Example 8D: discussion of activation energy calculation
The activation energy associated with water transport is assessed by measuring water permeability at different temperatures, and these energies can be used to determine whether transmembrane water transport is diffusion driven or channel mediated. Shrinkage experiments were performed on pristine liposomes and liposomes containing pR-PH channels (CLR 0.01) at different temperatures to generate an Arrhenius plot (Arrhenius plot; ln k versus 1/T) (FIG. 15E). The activation energy of the original liposome 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). The high activation energy suggests that water transport across the lipid bilayer is diffusion driven. After incorporation of pR-PH channels into liposomes, the activation energy was reduced to 7.77.+ -. 1.06kcal/mol, which is a strong evidence that water passage through pR-PH channels is channel-mediated. This value is comparable to the reported value of AQP0 mediated water activation energy (7.60±1.70 kcal/mol), with AQP0 being 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 CD absorbance signal at 306nm (FIGS. 18A and 18C). Thus, UV-vis and CD techniques are used to determine pR-PH channel efficiency for insertion into a liposome membrane (e.g., in the form of a liposome membrane), 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 channel (FIGS. 18B and 18D). The efficiency of insertion of the channels into liposomes, as determined by both techniques, exhibited similar values (48.13 ±7.76% UV-vis and 49.20 ±4.53% CD) when CLR was 0.005, as shown in fig. 18F. Based on the insertion efficiency of the channel and lipid, the single channel water permeability was calculated to be about 1.3x10 9 Water molecules/s/channel (FIG. 15G), which is comparable to the single channel water permeability of AQP 1 (4X 10) 9 Water molecules/s/channel) are equivalent (see also fig. 20).
Example 8F: discussion of solute rejection results
The reflectance has been used to estimate the relative trapping properties of the channels. The reflectance was determined based on a stopped-flow light scattering experiment by using different solutes as penetrants (fig. 19). Sucrose is chosen as the reference solute because of its relatively large molecular size. The reflectance was greater than 1 for all solutes (fig. 15F), indicating almost complete entrapment of these solutes. The retention properties are attributed to the narrow pores of pR-PH channels. The narrowest region has a diameter of about This is very close to the diameter of AQP1(see also fig. 20).
Example 8G: discussion of pR-PH channel comparison with other Water channels
The excellent properties of pR-PH channels allow us to compare it with biological aquaporins and previously reported artificial aquaporins (fig. 20). As a benchmark, aquaporin Z (AqpZ) was reconstituted into PC/PS liposomes in buffer, and the properties of AqpZ are also shown in FIG. 20. pR-PH channels were found to have the same 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 additional peptide column [5] arene in the contracted mode was two orders of magnitude lower than in the swelling experiment due to the increase in the low conductivity channels. Unlike the additional peptide column [5] arene, pR-PH channel showed similar water permeability values in the swelling and contraction modes suggesting the stability of water-conducting activity of pR-PH channel in the lipid bilayer membrane environment. In addition, another advantage of pR-PH channels over other artificial water channels based on column [5] aromatics without salt rejection is the excellent rejection of NaCl and small solutes, making them viable materials for incorporation into membrane materials for water purification applications.
Example 9: summary
The present invention and the examples described herein relate to the synthesis and characterization of (pR) -column [5] arenes as attachment peptides for high permeability and selectivity artificial water channels. The (pS) -and (pR) -isomers of the peptide-containing column [5] arenes have been synthesized and isolated.
One aspect of the invention provides methods for synthesizing (pS) -pillar [5] arenes and (pR) -pillar [5] arenes for attachment of peptides. In another aspect, (pR) -pillar [5] arenes are provided that act as attachment peptides for high permeability and selective water channels. The peptide-attached (pR) -pillar [5] arene can be incorporated into a liposome membrane. The water permeability and selectivity can be measured by a stopped-flow light scattering experiment. In another aspect, a UV-vis method is provided for determining the efficiency of insertion of peptide-attached (pR) -column [5] arene into a liposomal membrane (e.g., liposomal form). In yet another aspect, a Circular Dichroism (CD) method is provided that determines the insertion efficiency of (pR) -pillar [5] arene of an attachment peptide in a liposome membrane (e.g., a liposome).
While the present invention has been particularly shown and described with reference to particular embodiments, it will be understood by those skilled in the art that various changes in form and details 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 defined 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 (35)
1. A method of synthesizing a hydrolysate of peptide-attached column [5] arene, the method comprising:
forming a carboxyl group substituted column [5] arene from the dialkoxybenzene;
column substituted with said carboxyl group in organic solvent [5]]Aromatic hydrocarbon is mixed with tripeptide to form a column of attached peptide [5]]Aromatic hydrocarbons, wherein the tripeptide has a terminal-NH 2 A group and a terminal ester, wherein the tripeptide comprises NH 2 -L-Phe-L-Phe-L-Phe-O-methyl, NH 2 -L-Phe-L-Phe-L-Phe-O-ethyl, or NH 2 -L-Phe-L-Phe-L-Phe-O-propyl, wherein the peptide is attached to column [5]]Aromatic hydrocarbons include columns with the attachment peptides [5]]Diastereoisomers pR of aromatic hydrocarbons and columns of said attachment peptides [5]]Diastereomeric mixture of diastereomers of aromatic hydrocarbon pS, wherein said diastereomersConstruct pR has a different solubility in acetone than the diastereomer pS;
separating the diastereoisomer pR from the diastereoisomer pS based on the solubility difference in acetone; and
Hydrolyzing the diastereomer pR in the presence of a base to obtain a hydrolysate of the peptide-attached column [5] arene, wherein the hydrolysate of the peptide-attached column [5] arene consists of pR planar configuration.
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. The method according to claim 1 or 2, wherein the dialkoxybenzene comprises 1, 4-dimethoxybenzene.
4. The method of claim 2, wherein the organic acid catalyst comprises boron trifluoride diethyl etherate, p-toluene sulfonic acid, or trifluoroacetic acid.
5. The method of claim 2, wherein the dialkoxyl column [5] arene comprises 1, 4-dimethoxy column [5] arene.
6. The process according to claim 1 or 2, wherein the column [5] arene is represented by the formula:
7. the method of any one of claims 1-2, 5, 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, wherein the alkyl acetate comprises butyl bromoacetate, ethyl bromoacetate, methyl bromoacetate, or propyl bromoacetate.
10. The method of claim 7, wherein the carbonyl group substituted column [5] arene comprises an ethoxycarbonylmethoxy substituted column [5] arene.
11. The method of claim 7, 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 according to claim 1 or 2, wherein the carboxy group substituted column [5] arene is represented by the formula:
14. the method of any one of claims 1 to 2, 5, wherein mixing the carboxy-group substituted column [5] arene with the tripeptide comprises mixing the carboxy-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 according to any one of claims 1 to 2, 5, 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 2, 5, further comprising: subjecting the peptide-attached column [5] arene to column chromatography to produce a fraction comprising a diastereomeric mixture of the peptide-attached column [5] arene, mixing the diastereomeric mixture with acetone, and then filtering the diastereomeric mixture to obtain a residue and a filtrate, wherein the residue comprises diastereomer pR, and the filtrate comprises diastereomer pS.
17. The process according to any one of claims 1 to 2, 5, characterized in that the hydrolysis of the diastereoisomer pR comprises mixing the diastereoisomer pR with a base for 8 to 40 hours.
18. The method according to any one of claims 1 to 2, 5, wherein the base comprises lithium hydroxide, potassium hydroxide or sodium hydroxide.
19. A hydrolysate of peptide-attached column [5] arene configured as a water channel in a liposome membrane, wherein the hydrolysate of 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, wherein the terminal-COOH group extends away from the amide bond, and wherein the tripeptide comprises-NH-L-Phe-COOH, and wherein the hydrolysis product of a column [5] arene of the attachment peptide consists of pR planar configuration.
20. A method of synthesizing a liposome membrane comprising a hydrolysate of a peptide-attached column [5] arene, the method comprising:
synthesizing a hydrolysate of peptide-attached column [5] arene according to the method of any one of claims 1 to 18;
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, wherein the hydrolysis product of the peptide-attached column [5] arene consists of pR planar configuration.
21. The method of claim 20, wherein mixing the hydrolysate with the plurality of lipids comprises dissolving the hydrolysate in one or more organic solvents.
22. The method of claim 21, wherein the one or more organic solvents comprise chloroform and/or methanol.
23. The method of claim 22, wherein the chloroform and methanol are mixed at 0:1 to 20:1 by volume.
24. The method of any one of claims 20 to 23, wherein the plurality of lipids comprises phosphatidylcholine and/or phosphatidylserine.
25. The method of claim 24, wherein the phosphatidylcholine and/or phosphatidylserine is present at 0:1 to 100:1 is present in a molar ratio.
26. The method of any one of claims 20 to 23, wherein mixing the hydrolysate with the plurality of lipids comprises mixing the hydrolysate with the plurality of lipids in a molar ratio greater than 0 and up to 0.02.
27. The method of any one of claims 20 to 23, wherein the buffer comprises (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid), sodium chloride, and sodium azide.
28. The method of any one of claims 20 to 23, further comprising incubating the suspension at a temperature of 0 ℃ to 50 ℃ for 10 hours to 18 hours.
29. The method of any one of claims 20 to 23, wherein extruding the suspension comprises extruding the suspension more than once.
30. A liposome membrane comprising a plurality of lipids and a hydrolysate of at least one peptide-attached column [5] arene according to claim 19.
31. The liposome membrane of claim 30, wherein the plurality of lipids comprises phosphatidylcholine and/or phosphatidylserine.
32. The liposome membrane of claim 31, wherein the phosphatidylcholine and/or phosphatidylserine is present at 0:1 to 100:1 is present in a molar ratio.
33. The liposome membrane of any one of claims 30 to 32, wherein the hydrolysate and the plurality of lipids are present in a molar ratio of greater than 0 and up to 0.02.
34. The liposome membrane of any one of claims 30 to 32, wherein the liposome membrane comprises unilamellar liposomes.
35. The liposome membrane of claim 34, wherein the liposome has a diameter of 80nm to 250nm.
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