Receptor for the transport of zwitterions through organic membranes and a method of transport of zwitterions through organic membranes using said receptor

Abstract

A receptor for transport of zwitterions through organic membranes, characterised in that it has a sqaramide domain in one molecule, coordinating the anionic part of the zwitterion, an ether domain coordinating the cationic part of the zwitterion and a lipophilic domain, forming a molecular structure with the general formula (I) where the squaramide group is able to reversibly bind the anionic part of zwitterions by its coordination with amide group protons, an aryl group with an ether substituent (Ar-Y) is able to reversibly bind the cationic part of zwitterions by its coordination with free electron pairs of the oxygen atoms of the ether, an aryl group with a substituent (Ar-X) has lipophilic properties and imparts the lipophilicity of the receptor and the ability to dissolve it in organic membranes, wherein the receptor is lipophilic, displays affinity to the organic membrane and is soluble in organic solvents, mixtures of organic solvents and phospholipid membranes, and it is also able to form a complex with the zwitterion, soluble in organic solvents or mixtures of organic solvents, preferably immiscible with water, and in phospholipid membranes, wherein the receptor is able to extract zwitterions from the aqueous phase, with higher concentration of zwitterions, to the organic phase, preferably immiscible with water, and is also able to reverse extract the zwitterions from the organic phase, preferably immiscible with water, to the aqueous phase with lower concentration of zwitterions.The receptor according to the invention displays the ability to reversibly bind zwitterions dissolved in the aqueous phase, including amino acids of biological importance, as dipolar ions at physiological pH and displays the ability to transport them through organic membranes, also through phospholipid cell membranes. The receptor is inexpensive and easy to synthesise and it can potentially be applied in studies on the transport of amino acids through cell membranes in biologically active systems.

Description

[0001] RECEPTOR FOR THE TRANSPORT OF ZWITTERIONS THROUGH ORGANIC MEMBRANES AND A METHOD OF TRANSPORT OF ZWITTERIONS THROUGH ORGANIC MEMBRANES USING SAID RECEPTOR

[0002] The subject of the invention is a molecular receptor reversibly binding zwitterions (dipolar ions) present in the aqueous phase at physiological pH and capable of transporting them through organic membranes.

[0003] Bilayer lipid membranes (liposomes, membranes) are the necessary ingredients of living organisms and form spaces with separated chemical environments with different composition on both sides of the membrane. Specialised membrane proteins provide additional functionalities, such as molecular identification control and trans-membrane transport of ions, zwitterions and small molecules. Genetic defects in the natural carriers and protein channels are the cause of many conditions in humans [1-3], i.e. dysfunctions of congenital amino acid transporters have been related to various serious conditions, including neurodegenerative conditions, aminoaciduria, diabetes, obesity and cancer [4],

[0004] Intensive research dedicated to the development of a small molecule, synthetic transporter enabling the transport of ions through phospholipid cell membranes has been ongoing for several decades [5-13], The development of synthetic carriers and transmembrane channels capable of efficient and selective interactions with ions is one of the biggest challenges of supramolecular chemistry. A high number of efficient receptors able to transport chloride ions have been developed thus far [14-19], Most of these receptors have just a single function, i.e. they have one domain able to bind one ion type, although there are a few known ion-pair receptors

[0020] ,

[0005] A macrocyclic ion-pair receptor used in transmembrane transport is known

[0021] , This receptor is able to bind the ion pair within the macrocyclic cavity forming a complex and is also able to transport chloride ions in the presence of alkali metal cations. There are also known macrocyclic receptors based on calyx[4] pyrrole capable of selectively transporting caesium chloride through bilayer phospholipid membranes in the presence of other chloride salts [22,23], There are also known synthetic receptors incorporating aza-crown domains that bind cations, as well as cavities with three hydrogen bond donors that bind anions, functioning as M+ / O” symporters

[0024] ,

[0006] Reports about synthetic receptors used to transport zwitterions, such as amino acids, through lipid bilayers are extremely rare. Amino acid transport through organic membranes is very difficult, as at physiological pH (ca. 7.4) they are present as highly polar, dipolar ions (zwitterions) with high lipophobicity. There are known synthetic channels facilitating amino acid migration through lipid membranes, which are not molecular transporters [25-27], There is known a synthetic molecule which can transform into a zwitterion if exposed to UV radiation, able to transport phenylalanine

[0028] , There are also known amino acid transporters active under strictly defined conditions, in which amino acid transport is concerted with the transport of alkali metal cations [29-32], There is known a receptor for amino acid transport, which requires the polarity of the zwitterion to become decreased in a reversible reaction between the amino group of the amino acid and 3,5-bis-(trifluoromethyl)-benzaldehyde

[0033] , There is known a monofunctional, macrocyclic anion receptor based on calyxpyrrole, able to transport L-proline, however, it is ineffective in transporting more hydrophilic amino acids, such as glycine or alanine

[0034] , There are also known monofunctional anion receptors able to transport amino acids at physiological pH in their deprotonated, anionic form, which comprises 3% of the total amount of amino acids at neutral pH

[0035] , There are no reports available on receptors able to transport hydrophilic amino acids as zwitterions at physiological pH.

[0007] There are also known ion-pair receptors used to bind inorganic salts, however, these are not used to transport zwitterions through organic membranes. For example, there is known a macrocyclic ion-pair receptor able to bind and extract lithium bromide in solid-liquid extraction (SLE)

[0036] , There are also known macrocyclic ion-pair receptors able to extract lithium salts from the solid phase and from aqueous solutions [37-39], There are known tripodal receptors used to recognize lithium salts [40, 41], There is also known a receptor able to extract lithium chloride and lithium bromide from aqueous solutions to organic phase, where extracted lithium can be isolated as IJ2CO3 [42G], There are also known electrically neutral ion-pair receptors capable of simultaneously binding cations and anions, incorporating a squaramide moiety for anion binding and a crown ether unit for binding alkali metal cations. These receptors can coordinate ion pairs, including oxoanions and alkali metal cations, in particular sodium sulfate and potassium sulfate

[0043] , There are also known ion-pair receptors containing a squaramide group for binding anions and a crown ether group binding alkali metal cations, able to coordinate ion pairs in acetonitrile and to extract chloride salts from the aqueous phase to the organic phase

[0044] , There are also known neutral receptors containing a squaramide group for binding anions and a benzocrown group binding alkali metal cations, able to extract sodium chloride from the aqueous phase to nitrobenzene

[0045] , There are also known, squaramide-based, electrically neutral receptors for selective extraction of lithium halide from the solid phase to the liquid organic phase

[0046] ,

[0008] There is an unmet need to develop a non-toxic, easy to synthesis, inexpensive receptor able to achieve selective, efficient and reversible binding of zwitterions, especially amino acids in the form of zwitterions dissolved in water at physiological pH and their transport through organic membranes, particularly through phospholipid cell mebranes. There is also an unmet need to develop a protocol for the transport of zwitterions through organic membranes, which can find many medical uses in supported transport of amino acids through phospolipid cell membranes. This invention solves the problems known in the technical field.

[0009] Summary of the invention

[0010] A receptor for transport of zwitterions through organic membranes, characterised in that it has squaramide domain in one molecule, coordinating the anionic part of the zwitterion, an ether domain coordinating the cationic part of the zwitterion and a lipophilic domain, forming a molecular structure with the general formula: where the squaramide group is able to reversibly bind the anionic part of zwitterions by its coordination with amide group protons, an aryl group with an ether substituent (Ar-Y) is able to reversibly bind the cationic part of zwitterions by its coordination with free electron pairs of the oxygen atoms of the ether, an aryl group with a substituent (Ar-X) has lipophilic properties and imparts the lipophilicity of the receptor and the ability to dissolve it in organic membranes, wherein the receptor is lipophilic, displays affinity to the organic membrane and is soluble in organic solvents, mixtures of organic solvents and phospholipid membranes, and it is also able to form a complex with the zwitterion, soluble in organic solvents or mixtures of organic solvents, preferably immiscible with water, and in phospholipid membranes, wherein the receptor is able to extract zwitterions from the aqueous phase, with higher concentration of zwitterions, to the organic phase, preferably immiscible with water, and is also able to back extract the zwitterions from the organic phase, preferably immiscible with water, to the aqueous phase with lower concentration of zwitterions.

[0011] According to the invention, a crown substitute (Y) of the benzocrown group (Ar-Y) includes two alkoxyl substituents or a 9-crown-3 ether connected to a benzene ring, or a 12-crown-4 ether connected to a benzene ring, or a 15-crown-5 ether connected to a benzene ring, or a 18-crown-6 ether connected to a benzene ring. The aryl group with a substitutent (Ar-X) has electron acceptor properties while retaining the lipophilic nature, and preferably has substituents (X) such as: -F, -CF3, - NO2, -COOR, -CONR’R” or -SOiNR’R”, preferably -SOiNR’R” or -CONR’R”, where R, R’ and R” are aliphatic chains with 1-6 carbon atoms, aryl groups or hydrogen. The aryl group with a substituent (Ar-X) has electron donor properties or electron-neutral properties while retaining the lipophilic nature, it preferably has substituents (X) such as: -R or -OR, where R are aliphatic chains with 1-6 carbon atoms, aryl groups or H.

[0012] According to the invention, it is soluble in organic solvents immiscible with water, preferably in chloroform, dichloromethane, alkyl esters, aryl esters, alkyl ethers, aryl ethers, toluene and nitrotoluene, or in organic solvents miscible with water, preferably in acetonitrile, methanol, ethanol and 2-propanol, or in any liquid alcohol or a derivative thereof, or in mixtures of these solvents, and is also soluble in liposome membranes and is able to penetrate them. The receptor can be regenerated by washing the receptor or its solution with water, preferably clean water, most preferably with distilled water or deionised water, possibly with added methanol, followed by solvent evaporation.

[0013] The receptor preferably has one of the molecular structures I, II or III:

[0014] I II III

[0015] The method of zwitterion transport through organic membranes is characterised in that it uses the receptor described above, preferably a receptor with structure I, II or III, dissolved in an organic membrane in the form of an organic solvent, mixture of organic solvents or liposome membrane, said membrane is in contact with the aqueous source phase containing the zwitterion in its dipolar form, and is also in contact with the receiving aqueous phase, wherein the source phase and the receiving phase are separated, wherein the receptor reversibly binds the zwitterion by extracting it from the source phase, forming a complex soluble in the organic membrane, followed by release of the zwitterion in contact with the receiving phase, restoring the free receptor which remains in the organic membrane.

[0016] The process is preferably continuous, wherein the organic membrane containing the dissolved receptor remains simultaneously in contact with the source phase on one side and with the receiving phase on the other side, physically separating said phases, and the receptor continuously transports the zwitterion from the source phase to the receiving phase, wherein the receiving phase shows lower concentration of the zwitterion. Alternatively, the process is stationary and the following steps can be distinguished: step of zwitterion extraction from the source phase to the organic membrane, i.e. receptor solution saturation with the zwitterion, separation step, i.e. separation of the receptor complex solution containing the zwitterion from the source phase, and the secondary extraction step, i.e. decomposition of the receptor complex with the zwitterion resulting from the contact between the organic membrane and water, with zwitterion release to the aqueous phase.

[0017] According to the invention, the receptor is regenerated by washing with water, preferably clean water, most preferably distilled water or deionised water, possibly with added methanol, followed by solvent evaporation. The organic solvent or the mixture of organic solvents does not mix with water and forms two phases in contact with the aqueous solution, wherein the complex of the receptor and the zwitterion decomposes during the back extraction of the zwitterion to the aqueous phase, and the receptor remains dissolved in the organic phase, wherein the organic phase is preferably denser than the aqueous phase, the organic solvent or the mixture of organic solvents preferably contains chloroform, dichloromethane, alkyl ester, aryl ester, alkyl ether, aryl ether, toluene or nitrotoluene.

[0018] The receptor for zwitterion transport through organic membrane and the method of zwitterion transport through organic membranes using said receptor are described in detail below, in reference to the attached drawing, in which:

[0019] Fig. 1 presents the structural formula of the receptor according to the invention, with the general form of the aryl group with a substituent (ArX), with the general form of the benzocrown ether group (ArY);

[0020] Fig. 2 presents the structural formula of the receptor according to the invention, with the general form of the aryl group with a substituent (ArX) and with detailed structural variants of the benzocrown ether group (ArY), containing as substituent Y: alkoxy groups, 9-crown-3 ether, 12- crown-4 ether, 15-crown-5 ether or 18-crown-6 ether, where R are aliphatic chains with 1-6 carbon atoms;

[0021] Fig. 3 presents the structural formula of the receptor according to the invention with the general form of the benzocrown ether group (ArY) and with detailed structural variants of the aryl group with a substituent (ArX):

[0022] - with preferred electron accepting and lipophilic properties, containing the following as the X substituent: -F, -CF3, -NO2, -COOR, -CONR’R” or -SOiNR’R”, where the aryl group can have 1-5 X substituents, while R, R’ and R” are aliphatic chains with 1-6 carbon atoms, aryl groups or hydrogen,

[0023] - with electron-neutral or electron donor properties, i.e. -R or -OR, where R are aliphatic chains with 1-6 carbon atoms, aryl groups or hydrogen; Fig. 4 schematically presents the synthesis of the particularly preferred variant of receptor (I) and its structural formula;

[0024] Fig. 5 schematically presents the synthesis of a preferred variant of receptor (II) and its structural formula;

[0025] Fig. 6 schematically presents the synthesis of a preferred variant of receptor (III) and its structural formula;

[0026] Fig. 7 presents the1H NMR spectrum of the receptor in the preferred structural variant I, obtained in example 3;

[0027] Fig. 8 presents the13C NMR spectrum of the receptor in the preferred structural variant I, obtained in example 3;

[0028] Fig. 9 presents the1H NMR spectrum of the receptor in the preferred structural variant II, obtained in example 4;

[0029] Fig. 10 presents the13C NMR spectrum of the receptor in the preferred structural variant II, obtained in example 4;

[0030] Fig. 11 presents the1H NMR spectrum of the receptor in the preferred structural variant III, obtained in example 5;

[0031] Fig. 12 presents the13C NMR spectrum of the receptor in the preferred structural variant III, obtained in example 5;

[0032] Fig. 13 presents a summary of the1H NMR spectra of receptor I in deuterated chloroform (CDCh) during an experiment entailing amino acid extraction from aqueous solutions with physiological pH = 7.4, wherein the individual lines present: a) spectrum of a solution of free receptor I in wet CDCh, b) spectrum after extraction from an aqueous solution of glycine (50 mM), c) spectrum after extraction from an aqueous solution of -alanine (50 mM), d) spectrum after extraction from an aqueous solution of y-aminobutyric acid (50 mM), e) spectrum after extraction from an aqueous solution of 5-aminovaleric acid (50 mM), f) spectrum after extraction from an aqueous solution of LD-proline (50 mM), g) spectrum after extraction from an aqueous solution of L-ornitine (50 mM), h) spectrum after extraction from an aqueous solution of L-cysteine (5 mM), i) spectrum after back extraction of a receptor I solution (b-h) with deionised water, where the download shifts of the signals corresponding to squaramide proton (dot) compared to the spectrum of free receptor I can be seen (a) in the case of amino acid binding (b-h), wherein the spectrum of receptor I after back extraction (i) is identical to the spectrum of pure receptor I (a), which means complete release of the bound zwitterion;

[0033] Fig. 14 presents a summary of partial1H NMR spectra of receptor I at the concentration of 3 mmol / dm3in deuterated chloroform (CDCh) obtained during experiments entailing extraction from aqueous solutions, wherein the individual lines present: a) spectrum of a solution of free receptor I in wet CDCh, b) spectrum after extraction from an aqueous solution of glycine (50 mM), c) spectrum after extraction from an aqueous solution of NaCI (50 mM), d) spectrum after extraction from an aqueous solution of KCI (50 mM), e) spectrum after return extraction of a receptor I solution (b-d) with deionised water, where the download shifts of the signals corresponding to squaramide protons (dots) compared to the spectrum of free receptor I can be seen (a) in the case of alanine binding (a), in the case of alanine, NaCI and KCI binding (b-d), wherein the spectrum of receptor I after back extraction (e) is identical to the spectrum of pure receptor I (a), which means complete release of the bound zwitterion;

[0034] Fig. 15 schematically presents receptor I interaction with a zwitterion molecule (glycine) confirmed by 2D NMR ROESY measurements, where interaction directions designated as A, B, C were correlated with signals in 2D NMR spectra;

[0035] Fig. 16 schematically presents receptor II interaction with a zwitterion molecule (glycine) confirmed by 2D NMR ROESY measurements, where interaction directions designated as A, B were correlated with signals in 2D NMR spectra;

[0036] Fig. 17 presents the results of1H NMR experiments in D2O confirming transport of glycine transported by receptor I through the CHCh membrane, (source phase: 50 mM aqueous glycine solution; membrane: 5 mM solution of receptor I in CHCh, receiving phase: D2O); 3 mM aqueous solution of CH3COOK was used as the internal standard in each measurement, placed in a tightly closed mini-vial inside the NMR measurement vial, such that it did not mix with the studied glycine solution; the recorded1H NMR spectrum a) after 2 hours (2 mM glycine); b) after 6 hours (5 mM glycine); c) after 24 hours (10 mM glycine); d) after 48 hours (20 mM glycine) of the transport experiment; Fig. 18 presents a summary of13C NMR spectra of13C-1-glycine

[0037] - dissolved in a H2O / D2O mixture (9:1 v / v), and the individual lines present: a) spectrum of an aqueous solution of free glycine with one narrow signal, b) spectrum after addition of a paramagnetic Mn(NO3)2, which makes the signal of glycine wider,

[0038] - in the experiment of glycine transport into a POPC liposome, obtained in aqueous solution of HEPES (20 mM) with pH = 7.4, containing NaNOs (300 mM), Mn(NOs)2 (0.5 mM) and DMSO, where the individual lines present: c) spectrum of glycine solution introduced to the external solution, d) spectrum recorded 10 minutes after the addition of receptor I in DMSO to the external solution, wherein a narrow signal of free glycine transported by the receptor through organic membrane into the liposome appears after the addition of receptor I;

[0039] Fig. 19 presents a summary of19F NMR spectra of 3,3,3-trifluoro-DL-alanine

[0040] - dissolved in a H2O / D2O mixture (9:1 v / v), and the individual lines present: a) spectrum of an aqueous solution of free alanine with one narrow signal, b) spectrum after addition of a paramagnetic Mn(NO3)2, which makes the signal of alanine wider,

[0041] - in the experiment of glycine transport into a POPC liposome, obtained in aqueous solution of HEPES (20 mM) with pH = 7.4, containing NaNOs (300 mM), Mn(NOs)2 (0.5 mM) and DMSO, where the individual lines present: c) spectrum of alanine solution introduced to the external solution, d) spectrum recorded 10 minutes after the addition of receptor I in DMSO to the external solution, wherein a narrow signal of free alanine transported by the receptor through organic membrane into the liposome appears after the addition of receptor I;

[0042] Fig. 20 presents a summary of13C NMR spectra of13C-1 -glycine

[0043] - dissolved in a H2O / D2O mixture (9:1 v / v), and the individual lines present: a) spectrum of an aqueous solution of free glycine with one narrow signal, b) spectrum after addition of a paramagnetic Mn(NO3)2, which makes the signal of glycine wider,

[0044] - in the experiment of glycine transport into a POPC liposome, obtained in aqueous solution of HEPES (20 mM) with pH = 7.4, containing NaNOs (300 mM), Mn(NO3)2 (0.5 mM) and DMSO, where the individual lines present: c) spectrum of glycine solution introduced to the external solution, d) spectrum recorded 10 minutes after the addition of receptor III in DMSO to the external solution, wherein a narrow signal of free glycine transported by the receptor through organic membrane into the liposome appears after the addition of receptor III.

[0045] Detailed description of the invention

[0046] The invention is related to a receptor capable of reversibly binding zwitterions and transporting them through organic membranes, in particular of binding amino acids in the form of dipolar ions at physiological pH and their transport through phospholipid cell membranes. The invention is also related to the application of said receptor in zwitterion transport through organic membranes, in particular to transport amino acids as dipolar ions at physiological pH through phospholipid cell membranes.

[0047] The receptor according to the invention displays the ability to reversibly bind zwitterions dissolved in the aqueous phase, including amino acids of biological importance, as dipolar ions at physiological pH and displays the ability to transport them through organic membranes, also through bilayer (phospholipid) cell membranes. The receptor is inexpensive and easy to synthesise and it can potentially be applied in studies on the transport of amino acids through cell membranes in biologically active systems.

[0048] The receptor according to the invention is bifunctional, similarly to the recently obtained squaramide based ion-pair receptors [43-46], It was unexpectedly found that such receptors are not only capable of reversible binding and transporting (extracting) ion pairs, but also of binding and transporting (extracting) zwitterions, in particular of binding and transporting amino acids of biological importance through organic membranes. It is unexpected and surprising in that the distance between the anion a and cation binding domains in the receptor molecule is significantly larger than the distance between the anionic and cationic groups in biologically important amino acid molecules. Nevertheless, the strength of amino acid binding by the receptor according to the invention is high enough to allow amino acid extraction from water with the formation of a soluble complex of the receptor with the amino acid in the organic membrane (even in the case of hydrophilic amino acids, such as glycine) and amino acid release into the aqueous phase on the other side of said membrane (organic phase or bilayer cell membrane).

[0049] Receptor for transport of zwitterions from aqueous phase through organic membranes

[0050] The receptor according to the invention contains a group (domains) binding the anionic group of the zwitterion and a group (domain) binding the cationic group of the zwitterion, wherein the complex formed after the reversible binding of the zwitterion, similarly to the free receptor, is lipophilic and soluble in the organic phase in a wide range of concentrations, both in organic solvents and organic solvent mixtures miscible with water and immiscible with water, and is also soluble in liposome membranes. This enables the extraction of zwitterions from the aqueous phase to the organic phase and its back extraction to pure aqueous phase (for example, to deinionised water), as well as zwitterion transport through organic membranes from the first organic phase to the second organic phase.

[0051] Transport through organic membranes may be understood as two sequential processes: extraction from the first aqueous phase to the organic phase and the subsequent (secondary) extraction from the organic phase to the second aqueous phase, wherein the first aqueous phase contains the zwitterion in a higher concentration than the second aqueous phase and the difference between the concentrations of this zwitterion in both aqueous phases is the driving force behind the transport (transfer) of the zwitterion through the organic membrane. Transport through the membrane can also be understood as classical extraction from the aqueous phase to the organic phase and the secondary extraction to pure water (for example, to deionised water), then the membrane does not physically separate two aqueous solutions, but the nature of the extraction processes is identical to transport through a membrane physically separating two aqueous solutions. The membrane then actually separates the aqueous solutions, despite the fact that it does not simultaneously contact the solutions and contacts them only sequentially, but it acts as a connector between the solutions along the path of the zwitterion.

[0052] The general schematic design of the receptor according to the invention, containing an anion binding domain, a cation binding domain and a lipophilic domain is presented below: wherein the central group of the squaramide (amide domain) is capable of binding the anionic groups of the zwitterion by coordinating them with protons of the amide groups, the benzocrown ether group Ar-Y (ether domain) is capable of binding the cationic groups of the zwitterion by coordinating them with free electron pairs of oxygen atoms in the ether group, while the aryl group Ar-X increases the lipophilicity of the receptor, ensuring its solubility in the organic phase.

[0053] The receptor and its complexes with the zwitterion are soluble in the organic phase, in organic solvents or mixtures of organic solvents, as well as in liposome membranes, ensuring the possibility of extracting the zwitterion soluble in the aqueous phase to the organic phase, where a complex with the receptor is formed, which is of key importance from the point of view of enabling zwitterion transport through the organic membrane. The receptor is preferably soluble in chloroform, dichloromethane, alkyl esters, aryl esters, alkyl ethers, toluene and nitrotoluene or in their mixtures. The solubility of the receptor and of its complexes with the zwitterion in liposome membranes ensures its ability to pass through the phospholipid cell membranes and the potential, medical application of the receptor in amino acid transport into the cells of living organisms. The receptor and its complexes with the zwitterion are also soluble in organic solvents or mixtures of organic solvents miscible with water, preferably in acetonitrile, methanol, ethanol and 2-propanol or in any liquid alcohol or alcohol derivative, or in mixtures of such solvents.

[0054] According to the invention, the receptor and its complexes with the zwitterion are insoluble and unstable in water, which enables hydrolysis of the complex formed by the receptor with the zwitterion in contact with the aqueous phase with simultaneous, back extraction of the zwitterion to the aqueous phase. The free receptor remains in the organic phase during back extraction of the zwitterion to the aqueous phase. During transport through the organic membranes separating the two aqueous phases, the back extraction of the zwitterion occurs towards both aqueous solutions, but the process towards the first solution (source phase with high zwitterion concentration) is slower because of the equilibrium being shifted towards the complex formation with the receptor, while in the case of the second solution (receiving phase with low zwitterion concentration) this process is faster because of the equilibrium being shifted towards decomposition of the complex with the receptor, said decomposition being the driving force of the observed, macroscopic zwitterion transport through the membrane. On the other hand, during transport understood as separate processes of extraction from the aqueous phase (source phase) and back extraction to aqueous phase (receiving phase), preferably to deionised water, the driving force of these processes is the aim towards equilibrium between the receptorzwitterion complex and the free receptor and free zwitterion, which results in effective transport of zwitterions to the subsequent receiving phases if pure organic solution of the receptor and pure aqueous phase are used in the subsequent extraction stages. The squaramide unit (amide domain) is the domain binding the anionic groups of the zwitterion, where amide groups interact with the negatively charged anionic group of the zwitterion, preferably with the carboxylic group of the zwitterion. The strength of the carboxylic acid binding by the squaramide groups is additionally increased by the Ar-X and Ar-Y groups compared to the unsubstituted squaramide. Generally speaking, the Ar-X and Ar-Y groups decrease the electron density in the squaramide group, resulting in higher acidity of protons in its amide groups.

[0055] The ether group (Y) comprising part of the benzocrown ether group (Ar-Y) adjacent to the squaramide is the domain binding cationic groups, where the ether is connected to the squaramide via an aromatic ring. According to the invention, the ether group has from one to six oxygen atoms, corresponding to five possible variants of receptor structure, containing one of the following ether groups: alkoxy substituents -(OR)n, 9-crown-3 ether, 12-crown-4 ether, 15-crown-5 ether and 18-crown-6 ether, where R are alkyl chains with 1-6 carbon atoms, while n has value in the 1-5 range (Fig. 2).

[0056] The benzocrown ether group (Ar-Y) as such, when linked to the squaramide unit, does not impact the acidity of protons in the amide group. However, the strength of this interaction increases significantly after coordination of the cationic group of the zwitterion by the ether of the Ar-Y group. Thanks to this effect, the binding of the cationic group of the zwitterion increases the bond strength between the anionic group of the zwitterion and this receptor.

[0057] The aryl group with the lipid substituent (Ar-X) comprising a substituent of the squaramide group is intended to increase the lipophilicity of the entire receptor, thus facilitating solubility of this receptor and of its complex with the zwitterion in the organic phase. The substituents (X) should preferably increase the acidity of amide protons by withdrawing electron density from the aryl ring (i.e. the X substituent has electron acceptor properties), and thus from the squaramide group. This increases the capability of the squaramide groups to coordinate the anionic groups of the zwitterion, while retaining the lipophilic nature of the entire Ar-X group. The receptor preferably has (X) substituents, such as: -F, -CF3, -NO2, -COOR, -CONR’R” or -SOiNR’R”, most preferably -CONR’R”, where R, R’ and R” are aliphatic chains with 1-6 carbon atoms, aryl groups or hydrogen (Fig. 3).

[0058] It is also possible to use substituents (X) with electron donor properties (increasing electron density in the squaramide group) or electron-neutral properties (they do not change the electron density in the squaramide group), retaining lipophilic characteristics. For example, it is possible to use substituents (X) such as -R or -OR, where R are aliphatic chains with 1-6 carbon atoms, aryl groups or hydrogen (Fig. 3). Because of the decreased acidity of amide protons in the squaramide group (a receptor with electron donating X substituents) or because of the lack of impact on the acidity of these protons (a receptor with electron-neutral substituents X), such receptors are characterised by a lower zwitterion binding strength compared to receptors with substituents (X) with electron-accepting characteristics.

[0059] The aryl group (Ar-X) may have between 1 and 5 substituents (n = 1-5) in any positions and combinations (including combinations of electron-accepting and electron-donating or electron-neutral substituents), as the chemical nature of these substituents (electron acceptor properties) is much more important for the increased acidity of amide protons in the squaramide group than their positions. Nevertheless, the use of ArX groups is preferred: with five -F substituents (pentafluorophenyl group) or with two -CF3 substituents in meta positions, or with one -NO2 substitutent in the para position, or with two -COOR substituents in the meta position, or with one -SOiNR’R” substituent in the para position, or most preferably with two -OR substituents in the meta and para positions, or with two - CONR’R” substituents in the meta position.

[0060] According to the invention, a receptor for transport of zwitterions in aqueous phase through organic membranes preferably assumes one of the molecular structures I, II or III, where the aryl group (ArX) has two substituents -CONR’R’ in the meta position or two -OR substituents in the meta and para, while the benzocrown ether group (ArY) has an 18-crown-6 ether or two methoxy substituents:

[0061] During zwitterion binding by the receptor according to the invention, a complex soluble in the organic phase in a wide range of concentrations is formed. This enables the use of the receptor in zwitterion transport through organic membranes or in the extraction of zwitterions from the aqueous phase to the organic phase. It should be noted that every receptor has a slightly different selectivity and efficiency towards specific zwitterions.

[0062] The receptor according to the invention is less expensive and easier to synthesis than other known amino acid receptors. Additionally, it is the only one known receptor able to transport hydrophilic zwitterions in the form of dipolar ions at physiological pH (ca. 7.4). It allows the transport of zwitterions without the need to transform them into a cationic or an anionic form (acidic or basic pH). This allows the consideration of potential applications of the receptor to transport amino acids through cell membranes in living organisms at physiological pH, which may be of significant medical importance. The appropriate structure of the aryl group (Ar-X) provides the receptor and its complexes with the zwitterion lipophilic properties and solubility in organic membranes. The receptor may be incorporated in the phospholipid membrane (similar to membrane proteins) and comprise a synthetic mediator transporting zwitterions, including amino acids, through cell membranes of living organisms.

[0063] Method of zwitterion transport through organic membranes

[0064] The method of zwitterion transport through organic membranes according to the invention uses the receptor for zwitterion transport described above. Transport through organic membranes is possible thanks to the formation of a receptor complex after it reversibly binds the zwitterion, which remains soluble in a wide range of concentrations in organic solutions thanks to its structure and lipophilic properties, while it remains insoluble and unstable in water.

[0065] Transport through organic membranes may be understood as two sequential processes: extraction from the first aqueous phase to the organic phase, followed by the secondary extraction from the organic phase to the second aqueous phase, wherein the first aqueous phase contains the zwitterion in a higher concentration than the second aqueous phase and the difference between the concentrations of this zwitterion in both aqueous phases is the driving force behind the transfer of the zwitterion through the organic membrane. Transport through the membrane can also be understood as classical extraction from the aqueous phase to the organic phase and the back extraction to pure water (for example, to deionised water), then the membrane does not physically separate two aqueous solutions, but the nature of the extraction processes is identical to transport through a membrane physically separating two aqueous solutions. The membrane then actually separates the aqueous solutions, despite the fact that it does not simultaneously contact the solutions and contacts them only sequentially, but it acts as a connector between the solutions along the path of the zwitterion.

[0066] Continuous transport of zwitterions through organic membranes between two aqueous solutions or their extraction from the aqueous phase to the organic phase is possible and preferably concerted with secondary extraction to the second aqueous phase. An organic solvent or a mixture of organic solvents are used as the organic phase. The solvent must be selected such that the receptor complex with the zwitterion is well soluble in the solvent, otherwise the extraction is not going to be efficient. Upon contact of the source aqueous phase (with a higher zwitterion concentration) with the organic phase (containing the dissolved free receptor) at the interface, the receptor complex with the zwitterion is formed, followed by its transport (dissolution) into the organic phase (in the organic membrane). The affinity of the complex to the non-aqueous phase and its low initial concentration in this phase are the driving force behind this process. On the other hand, upon contact of the organic phase (containing the dissolved receptor-zwitterion complex) with the receiving aqueous phase (with a lower zwitterion concentration), hydrolysis (decomposition) of the recetpor-zwitterion complex occurs at the interface and the zwitterion is extracted to the second aqueous phase, while the free receptor remains in the organic phase. Zwitterion solubility in the aqueous phase is the driving force of this process.

[0067] As it was mentioned above, in the case of continuous transport through organic membranes separating two aqueous phases, the driving force of the entire process is the difference in rates between the secondary extraction of the zwitterion to the second aqueous phase (receiving phase), resulting from the lower zwitterion concentration compared to its concentration in the primary aqueous phase (source phase). On the other hand, in the case of transport understood as separate processes of extraction from the aqueous phase (source phase) and back extraction to aqueous phase (receiving phase), the driving force of these processes results from the drive towards equilibrium between the receptor-zwitterion complex and the free receptor and free zwitterion, which results in effective transport of zwitterions to the subsequent receiving phases if pure organic solution of the receptor and pure aqueous phase are used in secondary extraction.

[0068] The organic solvent or the organic solvent mixture are preferably immiscible with water and form two phases upon contact with the aqueous solution. The organic solvent or the organic solvent mixture immiscible with water contains chloroform, dichloromethane, alkyl esters, aryl esters, alkyl ethers, aryl ethers, toluene or nitrotoluene or their mixtures. Alternatively, the organic solvent or the mixtures of organic solvents mixes with water. In this variant, the organic solvent or the mixture of organic solvents preferably contains acetonitrile, methanol, ethanol, 2-propanol or any liquid alcohol or a derivative thereof, or mixtures of such solvents (this may find use in the process of zwitterion extraction from a solid to a liquid phase containing the receptor).

[0069] To ensure the ability to conveniently carry out continuous and sequential extraction, the organic phase immiscible with water is denser than the aqueous phase. This enables the membrane system to be formed in the so-called U-tube for continuous extraction or to carry out convenient sequential extraction using classical extractors.

[0070] According to the invention, transport through an organic membrane is carried out continuously, wherein the aqueous phase containing the zwitterion is located on one side of the organic membrane (for example, in one section of the U-tube, above the organic phase or outside the liposome), while the receiving aqueous phase is located on the other side of the organic membrane (for example, in the other section of the U-tube, above the organic phase or inside the liposome), wherein both aqueous phase remain in constant contact with said membrane. In the variant using the U-tube, the membrane is provided in the form of organic solution denser than the aqueous phase, which fills the bottom of the U-tube, with a depth at least equal to its thickness, preventing contact and mixing of both separated aqueous phases. In the variant using a liposome, the membrane has the form of a lipid bilayer separating the internal solution from the external solution. In this system, the receptor transports the zwitterion between the first and the second aqueous phases, while it remains in the organic phase.

[0071] Alternatively, transport through the organic membrane is stationary and the following steps may be distinguished in it: extraction from the source aqueous phase, separation of the organic phase and secondary (back) extraction to the receiving aqueous phase. The actual extraction involves saturation of the organic solution of the receptor with the zwitterion present in the source phase. According to the invention, a liquid sample containing the zwitterion is covered with an organic solution of the receptor and the system is then shaken or actively mixed through over the set duration, for example for 24 hours, to saturate the receptor solution. Next, the organic solution of the receptor and zwitterion complex is separated from the aqueous source phase, for example by decantation or using a separator. Next, secondary extraction to the receiving aqueous phase is carried out (preferably to deionised or distilled water) to separate the zwitterion and regenerate the receptor.

[0072] The zwitterion is separated through secondary extraction to the receiving aqueous phase, preferably to pure water of physiological pH in order to retain the dipolar nature of this molecule, most preferably to deionised or distilled water. At the interface of the organic phase and the aqueous phase, the complex decomposes and the zwitterion is transferred to the aqueous phase, while the free receptor remains in the organic phase. After potential separation of the receiving aqueous phase from the organic membrane, the zwitterion may be left in the aqueous phase for further steps or separated as a solid by evaporating water from the thus obtained aqueous solution.

[0073] The receptor can be regenerated by secondary extraction of the zwitterion to the aqueous phase, which is present in excess. The secondary extraction is carried out using water void of certain ions, preferably pure water, most preferably distilled water or deionised water. In the case of unfavourable coordination of the binding domains of the free receptor with water molecules in the organic phase, methanol is added to the organic solvent, preferably 5% of methanol by volume, which replaces water in the complex with the receptor by competing for the amide substituents. The solvent should be evaporated from the organic phase to separate the receptor as a solid.

[0074] The receptor for zwitterion transport in the aqueous phase through the organic membrane and the method of zwitterion transport through organic membranes using said receptor are described in detail below, in example embodiments. Example 1. Compounds M2 and M3 were obtained and then used to synthesise receptors with structure I and II. The synthesis was carried out according to the literature

[0047] , A solution (2.0 g, 6.4 mmol) of benzo-18-crown-6 ether in 50 ml of chloroform was added to a 100 mL flask, and the mixture was then cooled to 0°C. Concentrated nitric acid (10 mL), followed by 5 mL of acetic acid was added dropwise, while the solution was intensively mixed and low temperature was maintained. The mixture was kept at room temperature for 24 hours. Next, the suspension was diluted with chloroform (40 mL) and shaken twice with ice cold distilled water (60 mL). The organic phases were collected and dried over anhydrous MgSC Solvent evaporation yielded gel-like precipitate, which was washed with diethyl ether and stored in a freezer for 1 hour. The solid was filtered on a Schott funnel, washed with diethyl ether and dried under vacuum, obtaining compound M1 (4-nitrobenzo-18-crown-6 ether) (2.2 g, 6.2 mmol, yield 96%).

[0075] 1H NMR (300 MHz, DMSO-d6) 8 7.92-7.85 (d, 1H), 7.75 (s, 1 H), 7.22-7.15 (d, 1 H), 4.32-4.15 (s, 4H), 3.75-3.65 (s, 4H), 3.62-3.45 (m, 12H).

[0076] 13C NMR (75 MHz, DMSO-d6) 8 154.5, 148.3, 141.0, 118.1, 112.1, 107.7, 70.4, 70.3, 70.1, 69.3, 69.1, 68.8, 68.7.

[0077] HRMS (ESI) calculated for Ci6H23NO8Na [M + Na]+: 380.1321, observed: 380.1330.

[0078] 20 mg of 10% Pd / C was added to the degassed solution of 4-nitrobenzo-18-crown-6 ether (2.04 g, 5.72 mmol) in 60 mL of THF / MeOH (1:4) mixture. The reaction mixture was kept overnight in H2atmosphere (balloon pressure) at room temperature. The catalyst was removed by filtration through Celite and washed with MeOH. The filtrate was concentrated under vacuum, which resulted in the raw product M2 obtained with quantitative yield (1.87 g). The obtained 4-aminobenzo-18-crown ether (M2) was used in the next step without further purification.

[0079] To the solution of 4-aminobenzo-18-crown-6 ether (M2) (1.87 g, 5.72 mmol) in methanol (15 mL), 3,4- dimethoxy-3-cyxlobuteno-1,2-dion (0.81 g, 5.72 mmol) was added. The reaction mixture was kept at room temperature overnight. The obtained precipitate was filtered and washed with methanol. The product was purified using column chromatography on silica gel, using chloroform:methanol (9:1) as the eluent, and the M3 product was obtained as a white solid (1.68 g, yield 67%).

[0080] 1H NMR (300 MHz, DMSO-d6) 8 10.62 (s, 1 H), 7.2-6.7 (m, 3H), 4,36 (s, 3H), 4.12-3.98 (m, 4H), 3.82- 3.68 (m, 4H), 3.65-3.46 (m, 12H).

[0081] 13C NMR (75 MHz, DMSO) 8 189.24, 183.97, 178.73, 169.07, 148.80, 145.54, 131.86, 113.92, 112.10,

[0082] 106.56, 70.37, 70.34, 70.28, 69.21, 69.07, 68.88, 68.49, 60.93. HRMS (ESI): calculated for C2iH27NO9Na [M+ Na]+: 460.1584, observed: 460.1591.

[0083] Example 1. The compound TM were obtained and then used to synthesise receptors with structures I and III. To a solution of 5-nitroisophthalic acid (3.0 g, 14.2 mmol) in 20 mL of anhydrous dichloromethane cooled down to 2°C, oxalyl chloride (2.68 mL, 31.3 mmol) and dimethylformamide (41 mg, 0,57 mmol) were added dropwise under argon. The reaction was carried with the mixture being mixed for 2 hours at room temperature. Next, the reaction mixture was cooled down to 0°C and dibutylamine (4.8 mL, 28.5 mmol) and triethylamine (5.0 mL, 35,9 mmol) in anhydrous dichloromethane (20 mL) was added dropwise. The reaction mixture was kept at room temperature for 24 hours at room temperature, with stirring. Next, 20 mL of dichloromethane was added to the reaction mixture and shaken with 20 mL of 0.5 M HCI solution. The organic phase was separated and extracted with NaHCOs solution, and then dried using anhydrous MgSC The solved was then evaporated and the product was purified by column chromatography on silica gel (chloroform) and the TN compound was obtained as dark yellow oil (4.3 g, 9.94 mmol, yield 70%).

[0084] 1H NMR (300 MHz, DMSO-d6) 8 8.17 (s, 2H), 7.75 (s, 1 H), 3.55-3.35 (m, 4H), 3.20-3.05 (m, 4H), 1.70- 1.25 (m, 12H), 1.15-1.00 (m, 4H), 1.00-0.85 (m, 6H), 0.80-0.60 (m, 6).

[0085] 13C NMR (75 MHz, DMSO-d6) 8 167.80, 148.06, 139.46, 130.78, 121.97, 48.65, 44.56, 30.67, 29.60, 20.19, 19.60, 14.24, 13.81.

[0086] HRMS (ESI): calculated for C24H4oN304[M+H]+: 434.30133, observed: 434.30118.

[0087] The flask containing the solution of the TN compound (2.0 g, 4.6 mmol) in 20 mL of dry ethanol was placed in an oil bath kept at 70°C under argon and SnCI22 H2O (2.1 g, 9.3 mmol) was added. The reaction was continued for 1 hour and then left to cool and poured on ice. The mixture was then made alkaline up to pH ca. 10 by adding 5% aqueous NaOH solution. The obtained, basic mixture was mixed for one hour. The aqueous mixture was extracted four times with ethyl acetate, the organic phases were collected, washed with brine and dried using anhydrous MgSO4. The solvent was then evaporated and the product was dried in vacuum, resulting in the TA compound obtained as yellow oil (1.77 g, 4.4 mmol, yield 96%).

[0088] 1H NMR (300 MHz, DMSO-d6) 8 6.50 (s, 2H), 6.25 (s, 1 H), 5.44 (s, 2H), 3.45-3.35 (m, 4H), 3.30-3.05 (m, 4H), 1.60-1.30 (m, 8H), 1.40-1.20 (m, 4H), 1.20-1.00 (m, 4H), 1.00-0.85 (m, 6H), 0.85-0.60 (m, 6H).

[0089] 13C NMR (75 MHz, DMSO-d6) 8 170.78, 149.22, 138.49, 112.10, 111.19, 48.34, 43.87, 30.75, 29.67,

[0090] 20.11, 19.73, 14.25, 13.91. HRMS (ESI): calculated for C24H42N3O2 [M+H]+: 404.32715, observed: 404.32698.

[0091] To the solution of amine TA in methanol 3,4-dimethxy-3-cyclobuteno-1,2-dion (0.67 g, 4.7 mmol) and triethylamine (0.69 ml, 5 mmol) were added. The reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was then concentrated and the product was purified by column chromatography on silica gel (5% methanol in chloroform) and the TM compound was obtained as dark yellow solid (1.54 g, 3.0 mmol, yield 65%).

[0092] 1H NMR (300 MHz, DMSO-d6) 8 10.89 (s, 1 H), 7.38 (s, 2H), 6.92 (s, 1H), 4.39 (s, 3H), 3.75-3.30 (m, 4H), 3.25-2.95 (m, 4H), 1.7-1.20 (m, 12H), 1.15-0.95 (m, 4H), 0.95-0.80 (m, 6H), 0.80-0.55 (m, 6H).

[0093] 13C NMR (75 MHz, DMSO-d6) 8 188.14, 184.69, 184.57, 179.62, 169.49, 138.96, 138.62, 119.76, 117.72, 61.10, 48.39, 44.12, 30.61, 29.65, 20.08, 19.66, 14.25, 13.81.

[0094] HRMS (ESI): calculated for C29H44N3O5 [M+H]+: 514.32755, observed: 514.32725.

[0095] Example 3. The receptor with the structural formula I was obtained according to the following procedure (Fig. 4). To a solution of the M2 compound (0.44 g, 1.0 mmol) in methanol (10 mL) the TM compound (0.51 g, 1.0 mmol) and TEA (0.27 mL, 2.0 mmol) were added. After stirring for 24 hours at room temperature, the reaction mixture was concentrated and purified using column chromatography on silica gel (5% methanol in chloroform), thus obtaining receptor I as a yellow solid (0.63 g, 0.78 mmol, yield 78%).

[0096] The molecular structure of receptor I was confirmed with1H NMR measurements (300 MHz, DMSO- d6) 8: 9.93 (s, 1 H), 9.78 (s, 1 H), 7.46 (s, 2H), 7.27 (s, 1 H), 7.05-6.90 (m, 1 H), 6.90-6.75 (m, 2H), 4.20- 4.00 (m, 4H), 3.85-3.65 (m, 4H), 3.65-3.48 (m, 12H), 3.48-3.35 (m, 4H), 3.30-3.10 (m, 4H), 1.70-1.20 (m, 12H), 1.15-1.00 (m, 4H), 1.00-0.85 (m, 6H), 0.80-0.60 (m, 6H) (Fig. 7).

[0097] The molecular structure of receptor I was confirmed with13C NMR measurements (75 MHz, CDCh) 8: 182.83, 181.54, 169.56, 166.30, 165.18, 149.17, 145.14, 139.27, 139.17, 132.39, 118.89, 116.81, 114.37, 110.97, 105.51, 70.8-70.0 (m), 69.4-68.8 (m), 68.52, 48.47, 44.14, 30.66, 29.67, 20.09, 19.69, 14.29, 13.88 (Fig. 8).

[0098] The molecular structure of receptor I was confirmed with HRMS (ESI) measurements: calculated mass of the molecular peak for the molecule C44H64N40ioNa [M+Na]+: 831.45146, observed peak: 831.45162.

[0099] Example 4. The receptor with the structural formula II was obtained according to the following procedure (Fig. 5). To a degassed solution of 1,2-dibutoxy-4-nitrobenzene (DBN) (0.6 g, 2.24 mmol) in 20 mL of the THF / MeOH (1:4) mixture 10 mg 10% Pd / C was added. The reaction mixture was kept overnight in H2 atmosphere (balloon pressure) at room temperature. The catalyst was then removed by filtering through a Celite insert and washed with MeOH. The filtrate was concentrated under vacuum, which resulted in the raw product DBA obtained with quantitative yield (0.52 g). The obtained 3,4- dibutoxyaniline (DBA) was used in the next step without further purification. To a solution of the 3,4- dibutoxyaniline (0.27 g, 1.0 mmol) in methanol (10 mL) the M3 compound (0.5 g, 1.0 mmol) and TEA (0.31 mL, 2.20 mmol) were added. After the reaction was carried out for 24 hours at room temperature, the reaction mixture was concentrated and purified using column chromatography on silica gel (5% methanol in chloroform), obtaining receptor II as a dark green solid (0.51 g, 0.79 mmol, yield 69%).

[0100] The molecular structure of receptor II was confirmed with1H NMR measurements (300 MHz, DMSO- d6) 8: 10.50-10.30 (d, 2H), 7.50-7.35 (m, 2H), 7.05-6.85 (m, 4H), 4.20-4.05 (m, 4H), 4.05-3.95 (m, 2H), 3.95-3.87 (m, 2H), 3.85-3.70 (m, 4H), 3.70-3.45 (m, 12H), 1.80-1.60 (m, 4H), 1.55-1.35 (m, 4H), 1.05- 0.85 (m, 6H) (Fig. 9).

[0101] The molecular structure of receptor II was confirmed with13C NMR measurements (75 MHz, CDCh) 8: 181.43, 165.53, 165.45, 149.76, 148.61, 145.09, 144.37, 133.18, 132.91, 115.54, 113.30, 110.56, 110.38, 105.75, 105.58, 104.52, 70.00-67.79 (m), 31.43, 31.17, 19.22, 14.16 (Fig. 10).

[0102] The molecular structure of receptor II was confirmed with HRMS (ESI) measurements: calculated mass of the molecular peak for the molecule C34H46N2O10 [M-H+]_: 641.30797, observed peak: 641.30506.

[0103] Example 5. The receptor with the structural formula III was obtained according to the following procedure (Fig. 6). To a solution of 3,4-dimethoxyaniline (DMA) (0.15 g, 1.0 mmol) in methanol (10 mL) the TM compound (0.51 g, 1.0 mmol) and TEA (0.27 mL, 2.0 mmol) were added. After stirring for 24 hours at room temperature, the reaction mixture was concentrated and the product was purified using column chromatography on silica gel (5% methanol in chloroform), thus obtaining receptor III as a beige solid (0.44 g, 0.70 mmol, yield 70%).

[0104] The molecular structure of receptor III was confirmed with1H NMR measurements (300 MHz, DMSO- d6) 8:

[0105] 9.94 (s, 1 H), 9.79 (s, 1 H), 7.47 (s, 2H), 7.30 (s, 1 H), 7.00-6.80 (m, 3H), 3.90-3.65 (d, 6H), 3.55-3.35 (m, 4H), 3.30-3.05 (m, 4H), 1.65-1.25 (m, 12H), 1.20-1.00 (m, 4H), 1.00-0.85 (m, 6H), 0.85-0.60 (m, 6H) (Fig. 11).

[0106] The molecular structure of receptor II was confirmed with13C NMR measurements (75 MHz, CDCh) 8: 182.76, 181.46, 169.56, 166.26, 165.12, 149.77, 145.79, 139.31, 139.15, 132.37, 118.88, 116.79, 112.93, 110.70, 104.38, 56.21, 55.90, 48.47, 44.14, 30.67, 29.67, 20.10, 19.70, 14.24, 13.85 (Fig. 12). The molecular structure of receptor III was confirmed with HRMS (ESI) measurements: calculated mass of the molecular peak for the molecule C36H;iN4O6Na [M+Na]+: 635.38032, observed peak: 635.37995.

[0107] Example 6. A liquid-liquid extraction of the chloroform (CDCh) solution of receptor II (5 mM) was carried out with aqueous solution of one of the hydrophilic amino acids (glycine) (50 mM). Next, the organic layer was collected and subjected to 2D NMR experiments intended to determine the nature of interaction between the receptors and glycine (Fig. 15). ROESY NMR experiments on the chloroform solution after glycine extraction showed that the ammonium functional group of glycine is located near the benzocrown ether. On the other hand, the carboxylic anion interacts with the squaramide groups, as indicated by the shift of signals of these amide protons, respectively, from 9.00 ppm and 8.87 ppm to 9.86 ppm and 9.64 ppm. The middle part of glycine, namely the a protons, shows cross-peaks with aromatic protons and ethylene protons of the crown ether located near the aromatic ring. This data suggests simultaneous interaction of the cation and anion binding domains with the anionic and cationic part of the amino acid.

[0108] Example 7. A liquid-liquid extraction of the chloroform (CDCh) solution of receptor I (5 mM) was carried out with aqueous solution of one of the hydrophilic amino acids (glycine) (50 mM). Next, the organic layer was collected and subjected to 2D NMR experiments intended to determine the nature of interaction between the receptors and glycine (Fig. 16). ROESY NMR experiments on the chlorofom solution after glycine extraction showed that the ammonium functional group of glycine is located near the benzocrown ether. On the other hand, the carboxylate anion interacts with the squaramide groups, as indicated by the shift of signals of these amide protons, respectively, from 8.42 ppm and 8.66 ppm to 9.903 ppm and 9.642 ppm. The middle part of glycine, namely the a protons, shows cross-peaks with aromatic protons and ethylene protons of the crown ether located near the aromatic ring. This data suggests simultaneous interaction of the cation and anion binding domains with the anionic and cationic part of the amino acid.

[0109] Example 8. An experiment involving glycine transport from the aqueous source phase, through the organic chloroform (CHCh) membrane to the aqueous receiving phase using receptor I was carried out. 6 mL of receptor I solution in chloroform (5 mM) was poured into the U-tube, and this solution filled the bottom of both arms. Next, 6 mL of the aqueous solutions of the source phase and of the receiving phase were poured into each arm of the U-tube. An aqueous glycine solution (50 mM) was used as the source phase, with D2O used as the receiving phase. The increase of glycine concentration in the receiving phase was monitored using1H NMR measurements. The receiving solution was sampled after 2 h, 6 h, 24 h and 48 h. The measurements were carried out using coaxial NMR tubes with an insert containing an internal standard (CH3COOK). The analysed sample was immediately returned to the U- ' l. tube after each measurement and the experiment was continued. An increase in the glycine concentration in the receiving phase was observed in the subsequent measurements at 2 mM (2 h), 5 mM (6 h), 10 mM (24 h) and 20 mM (48 h), which proves that receptor I is capable of transporting the highly hydrophilic amino acids (glycine) in the form of a zwitterion. The obtained1H NMR spectra are summarised in Fig. 17.

[0110] Example 9. An experiment on the transport of labelled glycine through the lipid membrane into the POPC liposome using receptor I was carried out. In the 9:1 (v / v) H2O / D2O aqueous solution of the HEPES (20 mM) buffer with pH = 7.4, containing NaNOs (100 mM), POPC liposomes were formed (30 mM; 1,0 pm) according to the known literature recipe

[0048] , The external solution and the internal solution had identical compositions. Next, to the external solution, labelled glycine Gly-1-13C (50 mM) and Mn(NOs)2 (0,5 mM) were added to attenuate its signal. The13C NMR spectra showed a broader signal at ca. 173 ppm, originating from the complex of glycine with manganese(ll) ions (Fig. 18C). Next, a solution of receptor I in DMSO (10% mol) was added to the external solution. After 10 minutes, a narrow band was observed in13C NMR spectra, at ca. 173 ppm, originating from uncomplexed glycine (Fig. 18D). This provided proof of successful transport of free glycine molecules into the POPC liposome, where the complexating manganese(ll) ions were absent.13C NMR spectra obtained in this experiment were summarised in Fig. 18, together with reference spectra recorded for pure glycine solution (Fig. 18A) and solution of glycine not bound into a complex by manganese ions (Fig. 18B).

[0111] Example 10. An experiment on the transport of labelled alanine through the lipid membrane into the POPC liposome using receptor I was carried out. In the 9:1 (v / v) H2O / D2O aqueous solution of the HEPES (20 mM) buffer with pH = 7.4, containing NaNOs (100 mM), POPC liposomes were formed (30 mM; 1,0 pm) according to the known literature recipe

[0048] , The external solution and the internal solution had identical compositions. Next, to the external solution, fluorinated alanine 3,3,3-trifluoro- DL-alanine (50 mM) and Mn(NO3)2 (0,5 mM) were added to attenuate its signal. The19F NMR spectra showed a broader signal at ca. -71 ppm, originating from the complex of alanine with manganese(ll) ions (Fig. 19C). Next, a solution of receptor I in DMSO (10% mol) was added to the external solution. After 10 minutes, a narrow signal was observed in19F NMR spectra, at ca. -71 ppm, originating from uncomplexed glycine (Fig. 19D). This provided proof of successful transport of free alanine molecules into the POPC liposome, where the complexating manganese(ll) ions were absent.19F NMR spectra obtained in this experiment were summarised in Fig. 19, together with reference spectra recorded for pure alanine solution (Fig. 19A) and solution of alanine not bound into a complex by manganese ions (Fig. 19B). Example 11. An experiment involving transport of labelled glycine through the lipid membrane and into the POPC liposome using receptor III was conducted. In a 9:1 (v / v) H2O / D2O solution of the HEPES (20 mM) buffer with pH = 7.4, containing NaNOs (100 mM), POPC liposomes were formed (30 mM; 1.0 pm), according to the known literature recipe

[0048] , The external solution and the internal solution had identical compositions. Next, to the external solution, labelled glycine Gly-1-13C (50 mM) and Mn(NOs)2 (0,5 mM) were added to attenuate its signal. The13C NMR spectra showed a broader signal at ca. 173 ppm, originating from the complex of glycine with manganese(ll) ions (Fig. 20C). Next, a solution of receptor I in DMSO (10% mol) was added to the external solution. After 10 minutes, a narrow signal was observed in the13C NMR spectra, at ca. 173 ppm, originating from uncomplexed glycine (Fig. 20D). This provided proof of successful transport of free glycine molecules into the POPC liposome, where the complexating manganese(ll) ions were absent.13C NMR spectra obtained in this experiment were summarised in Fig. 20, together with reference spectra recorded for pure glycine solution (Fig. 20A) and solution of glycine not bound into a complex by manganese ions (Fig. 20B).

Claims

Claims1. A receptor for transport of zwitterions in the aqueous phase through organic membranes, characterised in that it has a squaramide domain in one molecule, coordinating the anionic part of the zwitterion, an ether domain coordinating the cationic part of the zwitterion and a lipophilic domain, forming a molecular structure with the general formula:- where the squaramide group is capable of reversibly binding the anionic part of zwitterions by its coordination with protons of the amide groups,- the aryl group with the ether substituent (Ar-Y) is capable of reversibly binding the cationic part of the zwitterions by its coordination with free electron pairs of oxygen atoms in the ether,- the aryl group with the substituent (Ar-X) has lipophilic properties and ensures lipophilicity of the receptor and its ability to dissolve in organic membranes,- the receptor has lipophilic properties, shows affinity to the organic membrane and is soluble in organic solvents, mixtures of organic solvents and phospholipid membranes,- the receptor can form a complex with the zwitterion, soluble in organic solvents or mixtures of organic solvents, preferably immiscible with water or mixtures of organic solvents,- the receptor can extract the zwitterions from the aqueous phase with higher zwitterion concentration to the organic phase, preferably immiscible with water,- the receptor can carry out back extraction of the zwitterions, from the organic phase, preferably immiscible with water, to aqueous phase with a lower zwitterion concentration.

2. A receptor according to Claim 1, characterised in that the crown substituent (Y) of the benzocrown group (Ar-Y) is- two alkoxy substituents, or- a 9-crown-3 ether connected to a benzene ring, or- a 12-crown-4 ether connected to a benzene ring, or- a 15-crown-5 ether connected to a benzene ring, or- a 18-crown-6 ether connected to a benzene ring.

3. A receptor according to Claim 1 or 2, characterised in that the aryl group with a substitutent (Ar-X) has electron acceptor properties while retaining the lipophilic nature, and preferably has substituents (X) such as: -F, -CF3, -NO2, -COOR, -CONR’R” or -SOiNR’R”, preferably -SOiNR’R” or -CONR’R”, where R, R’ and R” are aliphatic chains with 1-6 carbon atoms, aryl groups or hydrogen.

4. A receptor according to Claim 1 or 2, characterised in that the aryl group with a substitutent (Ar-X) has electron donor or electron-neutral properties while retaining the lipophilic nature, and preferably has substituents (X) such as: -R or -OR, where R are aliphatic chains with 1-6 carbon atoms, aryl groups or hydrogen.

5. A receptor according to any of the Claims 1-4, characterised in that it is soluble in organic solvents immiscible with water, preferably in chloroform, dichloromethane, alkyl esters, aryl esters, alkyl ethers, aryl ethers, toluene and nitrotoluene, or in organic solvents miscible with water, preferably in acetonitrile, methanol, ethanol and 2-propanol, or in any liquid alcohol or a derivative thereof, or in mixtures of these solvents, and is also soluble in liposome membranes and is able to penetrate them.

6. A receptor according to any of the Claims 1-5, characterised in that it can be regenerated by washing the receptor or its solution with water, preferably clean water, most preferably with distilled water or deionised water, possibly with added methanol, followed by solvent evaporation.

7. A receptor according to any of the Claims 1-6, characterised in that the receptor assumes one of the molecular structures I, II or III:

8. A method of zwitterion transport through organic membranes, characterised in that it uses the receptor described in Claims 1-7, preferably a receptor with structure I, II or III, dissolved in an organic membrane in the form of an organic solvent, mixture of organic solvents or liposome membrane, said membrane is in contact with the aqueous source phase containing the zwitterion in its dipolar form, and is also in contact with the receiving aqueous phase, wherein the source phase and the receiving phase are separated, this receptor reversibly binds the zwitterion by extracting it from the source phase, forming a complex soluble in the organic membrane, followed by release ofthe zwitterion in contact with the receiving phase, restoring the free receptor which remains in the organic membrane.

9. A method according to Claim 8, characterised in that the process is carried out continuously, wherein the organic membrane containing the dissolved receptor remains simultaneously in contact with the source phase on one side and with the receiving phase on the other side, physically separating said phases, and the receptor continuously transports the zwitterion from the source phase to the receiving phase, wherein the receiving phase shows lower concentration of the zwitterion.10.A method according to Claim 7, characterised in that the process is stationary and the following steps can be, distinguished in it:- the step of zwitterion extraction from the source phase to the organic phase, i.e. saturation of the receptor solution with the zwitterion,- the separation step, i.e. separation of the receptor complex solution containing the zwitterion from the source phase,- the secondary extraction step. i.e. decomposition of the receptor-zwitterion complex as a result of the contact between the organic membrane and water with the zwitterion being returned to aqueous phase.

11. A method according to Claim 7, characterised in that the receptor is regenerated by washing with water, preferably clean water, most preferably distilled water or deionised water, possibly with added methanol, followed by solvent evaporation.

12. A method according to Claim 7, characterised in that the organic solvent or the mixture of organic solvents does not mix with water and forms two phases in contact with the aqueous solution, wherein the complex of the receptor and the zwitterion decomposes during the back extraction of the zwitterion to the aqueous phase, and the receptor remains dissolved in the organic phase, wherein the organic phase is preferably denser than the aqueous phase, the organic solvent or the mixture of organic solvents preferably contains chloroform, dichloromethane, alkyl ester, aryl ester, alkyl ether, aryl ether, toluene or nitrotoluene.

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