Transmembrane pore formed by aromatic oligoamide foldings and uses thereof
阅读说明：本技术 通过芳香族低聚酰胺折叠体形成的跨膜孔及其用途 (Transmembrane pore formed by aromatic oligoamide foldings and uses thereof ) 是由 龚兵 于 2018-11-15 设计创作，主要内容包括：提供了芳香族低聚酰胺折叠体及其自组装组合物。芳香族低聚酰胺折叠体和组合物可形成可在膜中形成孔的管状结构。孔可用于运输离子和分子(例如冷冻保护剂或治疗剂)通过膜。管状结构在低温下表现出期望的稳定性。(Aromatic oligoamide folds and self-assembling compositions thereof are provided. The aromatic oligoamide folds and compositions can form tubular structures that can form pores in the film. Pores may be used to transport ions and molecules (e.g., cryoprotectants or therapeutic agents) through the membrane. The tubular structure exhibits the desired stability at low temperatures.)
1. A compound having the structure:
wherein R and R' are independently at each occurrence selected from the group consisting of a straight chain alkyl group, a branched chain alkyl group, an ether group, and an oligoether group;
x is acyl;
y is:
i)-NHCH3、-NHCH2CH3-NHR', and-NHAr; or
ii)-OCH3、-OCH2CH3、-OCH2CH3、-OC(CH3)3and-OR ",
wherein Ar is aryl and R' is a linear or branched alkyl; and is
n is 1 to 64.
2. The compound of claim 1, wherein R and R' are independently at each occurrence selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, and isopentyl.
3. The compound of claim 1, wherein R and R' are independently selected at each occurrence from Wherein the asterisk indicates stereochemistry R or S form, n is 1, 2, 3, 4, 5 or 6, and R' "is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentylAnd isoamyl.
4. The compound of claim 1, wherein the backbone of the compound is folded such that a helix is formed that extends longitudinally in the direction of the longitudinal axis, wherein the helix has a left-handed or right-handed orientation.
5. The compound of claim 4, wherein the helix has about 6.5 residues per turn.
6. The compound of claim 4, wherein said helix has about per turnThe pitch of the thread.
7. The compound of claim 4, wherein the helix has an interior and an exterior.
8. The compound of claim 7, wherein said interior is a hollow tubular cavity parallel to said longitudinal axis.
9. The compound of claim 7, wherein the inner diameter of the interior is from 7 to
10. The compound of claim 1, wherein the compound has a length along the longitudinal axis of from 3.5 to
11. The compound of claim 7, wherein the interior is hydrophilic.
12. A spiral composition comprising an assembly of identical compounds according to claim 1 or an assembly of different mixtures of compounds according to claim 1, wherein each of the identical compounds or each of the different mixtures of compounds is disposed on adjacent compounds to form a cylindrical structure such that the longitudinal axes of each compound are coaxially aligned, wherein the cylindrical structure has an exterior and an interior.
13. The spiral composition of claim 12, wherein the interior is a continuous hollow tubular cavity.
14. The spiral composition of claim 12, wherein the spiral composition has a length along the longitudinal axis of from 3.5 to
15. A method of using a compound according to claim 1 and/or a helical composition according to claim 11, comprising contacting a compound according to claim 1 and/or a helical composition according to claim 11 with a vesicle having a membrane, wherein the compound and/or the helical composition forms pores in the membrane.
16. The method of claim 15, wherein the membrane is a plasma membrane or a cell membrane.
17. The method of claim 15, further comprising transporting a target molecule through the pore.
18. The method of claim 15, wherein said contacting comprises administering said compound and/or said composition to an individual in need of treatment.
19. The method of claim 18, wherein the method further comprises administering a molecule of interest to the individual in need of treatment.
20. The method of claim 19, wherein the target molecule is a hydrophilic compound and/or a hydrophilic species.
21. The method of claim 20, wherein the hydrophilic compound and/or the hydrophilic material is a carbohydrate, a polyol, or a combination thereof.
22. The method of claim 21, wherein the carbohydrate is selected from the group consisting of glucose, sucrose, trehalose, glycerol, and sorbitol.
23. The method of claim 20, wherein the hydrophilic compound and/or the hydrophilic species are selected from protons, ions, dyes, peptides, CPA, drugs, adjuvants, hydrophilic chelators of metal ions, and combinations thereof.
24. The method of claim 23, wherein the CPA is an antifreeze peptide, a non-natural antifreeze oligomer, or a combination thereof.
25. The method of claim 15, wherein the method is performed in vivo, in vitro, or ex vivo.
26. A composition comprising at least one compound forming a continuous hollow tubular structure for forming pores in a membrane, wherein the pores are stable at low temperatures but are disrupted at elevated temperatures due to thermal motion.
27. The composition of claim 26, wherein the pores are stable at temperatures of 1 to 37 ℃.
28. The composition of claim 26, further comprising a plurality of the same or different compounds.
29. The composition of claim 28, wherein a plurality of compounds self-assemble into a supramolecular structure, wherein each compound is disposed on an adjacent compound to form a cylindrical structure such that the longitudinal axes of each compound are coaxially aligned, wherein the cylindrical structure has an exterior and an interior.
30. The composition of claim 26, wherein the at least one compound is a helix extending longitudinally in the direction of the longitudinal axis.
31. The composition of claim 30, wherein the helix is dextrorotatory or levorotatory.
32. The composition of claim 30, wherein the helix has about 6.5 residues per turn.
33. The composition of claim 30, wherein the helix has about each turnThe pitch of the thread.
34. The composition of claim 30, wherein the composition has a length along the longitudinal axis of from 3.5 to
35. The composition of claim 30, wherein the tubular structure has an interior and an exterior.
36. The composition of claim 35, wherein the interior is hydrophilic.
37. The composition of claim 35, wherein the interior is a hollow tubular cavity parallel to the longitudinal axis.
38. The composition of claim 35, wherein the inner diameter of the inner portion is from 7 to
39. The composition of claim 35, wherein the exterior of the helix has one or more hydrogen bonds.
Technical Field
The present disclosure relates generally to oligomeric amide folds. More particularly, the present disclosure relates to self-assembled aromatic oligoamide folds that can be used to form transmembrane pores.
Background
The plasma membrane plays an important biological role by acting as an osmotic barrier that prevents unassisted movement of most water-soluble substances. This membrane maintains the difference in properties between the inside and the outside of the cell. In biological systems, the permeability of cell membranes is regulated by passive pores driven along a concentration gradient and active transport proteins against a concentration gradient.
Efforts to establish synthetic channels began thirty years ago and were still in progress, with most systems based on ion transport and few on molecular transport. Synthetic organic pores have unique advantages over protein pores, including significantly smaller molecular mass, synthetic tunability (which allows for the introduction of building blocks that are incompatible with or cannot be introduced into protein-based pores), high stability, and non-immunogenicity. However, due to the limitations of current synthetic methods, the structural variability of membrane proteins has not been widely exploited and synthetic constructs are difficult to achieve. Indeed, functional macropores that can be readily synthesized with minimal biological methods are rare.
The use of native pore-forming proteins for intracellular delivery of hydrophilic molecules (e.g., sugars) has shown promise, it has previously been demonstrated that intracellular delivery of 0.2M trehalose can significantly improve the viability of cells after thawing when transported by genetically engineered variants of pore-forming toxin α -hemolysin α -the large lumen of hemolysinSufficient transport of molecules such as trehalose can be allowed, but since the macropores remain open, especially at physiological temperatures, cytotoxicity based on lack of selective transport replaces the unfavorable CPA cytotoxicity. Reasonable blocking strategies are crucial to reduce toxicity and by adding high concentrationsZn of degree2+In the past few decades, many chemists have been attracted to overcome the deficiencies of protein pores by developing synthetic pores capable of mimicking natural systems, such as synthetic efficiency and structural diversity to engineer a variety of functions, such as responsiveness and selective transport.
Disclosure of Invention
The present disclosure provides aromatic oligoamide folds and self-assembled compositions formed from aromatic oligoamide folds. The compounds and compositions can form tubular structures that can form pores in the film. The present disclosure also provides uses of the compounds and compositions of the present disclosure.
In one aspect, the present disclosure provides compounds comprising a folded oligoamide (also referred to herein as a "folder"). The oligomeric amide comprises a plurality of aromatic substituents connected by at least one amide bond.
In one example, the compounds of the present disclosure have a curved backbone. Without being bound by any particular theory, the curved backbone is primarily due to intramolecular hydrogen bonds, which rigidify the amide bond of each amide group with each aromatic substituent, and is at least partially due to interactions between the aromatic substituents (e.g., pi-pi interactions), thereby stabilizing the curved backbone.
In various examples, the aromatic substituents of the present disclosure have the following structure:
(hereinafter referred to as "B", "N", and "BN" residues, respectively), wherein R and R' are independently selected from the group consisting of: straight chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and the like), branched alkyl groups (e.g., branched derivatives of propyl, butyl, pentyl, hexyl, heptyl, octyl, and the like), and ether and oligoether groups (e.g., - (CH)2)2OCH3、-(CH2)2OCH2CH3、-(CH2)2OCH2CH(CH3)2、-(CH2)2O(CH2)2CH(CH3)2Etc.); x is an acyl group (e.g., acetyl, trifluoroacetyl, phenylacetyl, fluorenylmethoxycarbonyl, etc.) or an aryl substituent; and Y is i) -NHCH3、-NHCH2CH3-NHR "or-NHAr, ii) -OCH3、-OCH2CH3、-OCH2CH3、-OC(CH3)3OR OR 'where Ar is aryl and R' is a straight OR branched chain alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, etc.), OR an aromatic substituent.
Other examples of oligoether groups include, but are not limited toEtc., wherein the asterisks indicate a stereogenic carbon (i.e., a carbon having a stereochemical R-type or S-type), n is 1, 2, 3, 4, 5, or 6, and R' "is a linear or branched alkyl group (e.g., methyl, ethyl, propyl, isopropyl, etc.).
In one example, the compounds of the present disclosure form a folded tubular structure (e.g., a helix). In one non-limiting illustrative example, the compound folds as shown in the following structure (using B aromatic substituents as examples, and not excluding N, BN and its combinations (including those with B)):
where starting from the bold end of the structure moving clockwise, the compound spirals down into the page.
In one aspect, the present disclosure provides compositions comprising compounds of the present disclosure. In one example, a plurality of compounds of the present disclosure are assembled such that a plurality of the compounds are stacked on top of each other to form a cylindrical structure. The cylindrical structure is a composite of assembled compounds, and the longitudinal axes of each compound are coaxially aligned. The cylindrical structure has an interior and an exterior. The interior of the cylindrical structure is a continuous hollow tubular cavity. Other assemblies are contemplated and are within the scope of the invention.
The compositions of the present disclosure may form a helix (and thus may be referred to as a helix or a spiral composition). The helix may be right-handed or left-handed.
In one aspect, the present disclosure provides the use of compounds and/or compositions of the present disclosure. In various examples, one or more compounds and/or compositions of the disclosure are used to form pores in vesicles (e.g., to form transmembrane pores in liposomes and/or cells). The pores may be nanopores. In various examples, the compounds and/or compositions of the present disclosure can be administered to a cell, tissue, organ, or individual (e.g., an individual in need thereof). The subject may be a human, a non-human mammal, a non-mammal, or a plant.
The compounds and/or compositions of the present disclosure may form transmembrane pores. For example, the membrane is a membrane of a liposome, cell, or other similar vesicle or molecular system. The compounds and/or compositions of the present disclosure may be referred to as pore-forming compounds and/or compositions.
Target molecules can be transported through pores (e.g., transmembrane pores) formed by the compounds and/or compositions of the present disclosure. In many instances, the target molecule is a hydrophilic species. Thus, in various examples, the methods of the present disclosure include contacting a membrane of a vesicle (e.g., a liposome and/or a cell or similar system) having a transmembrane pore with a target molecule, where the target molecule is transported into the vesicle (e.g., a liposome and/or a cell or similar system). In various additional examples, a molecule of interest (e.g., a hydrophilic species) is transported out of a cell. Non-limiting examples of target molecules include ions (e.g., protons, sodium, potassium, or chloride ions), dyes (e.g., 8-hydroxypyrene-1, 3, 6-trisulfonate trisodium salt (HPTS)), nutrients commonly used in cell or tissue culture, small molecules (e.g., drugs, such as anticancer drugs, 5-fluoro-2' -deoxyuridine (5-FdU), etc.), carbohydrates (e.g., pentoses and hexoses, such as, but not limited to, mannose, glucose, galactose, etc.; disaccharides, such as, but not limited to, sucrose, trehalose, etc.); polyols (also referred to herein as "sugar alcohols," such as mannitol, sorbitol, and the like), chelating agents for metal ions (e.g., hydrophilic chelating agents for metal ions, such as hydrophilic chelating agents for fe (ii) and/or fe (iii)), cryopreservatives (CPAs, such as, but not limited to DMSO, ethylene glycol, and propylene glycol), peptides, and combinations thereof. Examples of suitable CPAs, ions and nutrients are known in the art.
Methods of using the compounds and/or compositions of the present disclosure include: i) contacting the membrane of a vesicle (e.g., a liposome, a cell, or a similar vesicle or molecular system) with at least one compound and/or composition of the present disclosure such that the compound and/or composition forms pores in the membrane of the vesicle, wherein the vesicle optionally encapsulates a stimulus-responsive molecule (e.g., a fluorescent dye); ii) contacting an analyte (e.g., a solution comprising an analyte, such as a carbohydrate, peptide, dye, and/or ion) with the vesicle such that the analyte interacts (e.g., binds) with the pore; and iii) measuring a change in fluorescence emission of the encapsulated stimulus-responsive molecule.
In one aspect, the compounds and/or compositions of the present disclosure may be used for molecular scale chromatography.
Drawings
For a fuller understanding of the nature and objects of the present disclosure, reference should be made to the following detailed description taken together with the accompanying figures.
Figure 1 shows three series of aromatic oligoamides folded into a helical conformation containing internal pores of different diameters.
Fig. 2 shows the energy-minimized folded structures of (a) 8-mer, 16-mer, 32-mer and (B) 64-mer of B-series oligomers in which all side chains (R groups) are substituted with methyl groups. The approximate length of each helix is shown.
FIG. 3 shows the general procedure for the synthesis of series B oligoamides. The same synthetic procedure is equally applicable to the preparation of N and BN series oligoamides.
Figure 4 shows the proton transport activity of B series oligomeric amide foldings.
Detailed Description
The claimed subject matter is now described with respect to only certain examples, but other examples, including examples that do not provide all of the advantages and features set forth herein, are also within the scope of this disclosure. Various structural, logical, process step, and electrical changes may be made without departing from the scope of the present disclosure.
Ranges of values are disclosed herein. The ranges set forth below include the lower and upper limits. Unless otherwise indicated, this range includes all values up to the order of the minimum value (lower or upper) and ranges between values in the range.
As used herein, unless otherwise specified, the term "group" refers to a chemical entity having one end that can be covalently bonded to other chemical species. Examples of groups include, but are not limited to:and
as used herein, unless otherwise specified, the term "moiety" refers to a chemical entity having two or more ends that can be covalently bonded to other chemical species. Examples of moieties include, but are not limited to:
as used herein, unless otherwise specified, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl, and the like. For example, the alkyl group may be C1To C12Alkyl, including all integer carbon numbers and carbon number ranges (C) therebetween1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11And C12). Alkyl groups may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, various substituents such as halogens (-F, -Cl, -Br, and-I), aliphatic groups (e.g., alkyl, alkenyl, alkynyl), aryl groups, alcohol/phenoxide groups (alkoxide groups), carboxylate groups (carboxylates groups), carboxylic acids, ether groups, amines, alcohols, thiols, and the like, and combinations thereof.
As used herein, unless otherwise specified, the term "aryl" refers to C5To C14Including all integer carbon numbers and carbon number ranges (C) therebetween5、C6、C7、C8、C9、C10、C11、C12、C13And C14). The aryl groups can comprise polyaryl moieties, such as fused ring or biaryl moieties. Aryl groups may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, various substituents such as halogens (-F, -C, -Br, and-I), aliphatic groups (e.g., alkenes, alkynes), aryl groups, alcohol/phenoxides, carboxylates, carboxylic acids, ether groups, amines, alcohols, thiols, and the like, and combinations thereof. Examples of aryl groups include, but are not limited to, phenyl, biaryl (e.g., biphenyl), and fused ring groups (e.g., naphthyl).
The compounds and compositions of the invention may be used for cryoprotection, nanopore-facilitated transport and/or uptake (or drug delivery) of therapeutic molecules, and/or other pharmaceutical or biological applications. For example, compounds and compositions may be used for drug delivery by releasing the contents of a capsule (e.g., liposome), for biotherapeutic by permeabilizing cells to cytotoxic drugs, or for biological preservation by loading cells with bioprotective agents.
The present disclosure provides aromatic oligoamide folds and self-assembled compositions formed from aromatic oligoamide folds. The compounds and compositions can form tubular structures that can form pores in the film. The present disclosure also provides uses of the compounds and compositions of the present disclosure.
In one aspect, the present disclosure provides compounds comprising a folded oligoamide (also referred to herein as a "folder"). The oligomeric amide comprises a plurality of aromatic substituents connected by at least one amide bond.
In one example, the compounds of the present disclosure have a curved backbone. Without being bound by any particular theory, the curved backbone is primarily due to intramolecular hydrogen bonds, which rigidify the amide bond of each amide group with each aromatic substituent, and is at least partially due to interactions between the aromatic substituents (e.g., pi-pi interactions), thereby stabilizing the curved backbone.
In various examples, the aromatic substituents of the present disclosure have the following structure:
(hereinafter referred to as "B", "N", and "BN" residues, respectively), wherein R and R' are independently selected from the group consisting of: straight chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and the like), branched alkyl groups (e.g., branched derivatives of propyl, butyl, pentyl, hexyl, heptyl, octyl, and the like), and ether and oligoether groups (e.g., - (CH)2)2OCH3、-(CH2)2OCH2CH3、-(CH2)2OCH2CH(CH3)2、-(CH2)2O(CH2)2CH(CH3)2Etc.); x is an acyl group (e.g., acetyl, trifluoroacetyl, phenylacetyl, fluorenylmethoxycarbonyl, etc.) or an aryl substituent; and Y is i) -NHCH3、-NHCH2CH3-NHR "or-NHAr, ii) -OCH3、-OCH2CH3、-OCH2CH3、-OC(CH3)3OR OR 'where Ar is aryl and R' is a straight OR branched chain alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, etc.), OR an aromatic substituent.
Other examples of oligoether groups include, but are not limited to
The compounds may have a variety of numbers of aromatic substituents (e.g., aryl moieties such as B, N and BN residues). In one example, the compound has 1 to 128 aromatic substituents, including all integers. In various examples, the compound is an 8-mer, 10-mer, 12-mer, 16-mer, 32-mer, 64-mer, or 128-mer, where the integer (e.g., 8, 16, etc.) corresponds to the number of aromatic substituents (e.g., aryl moieties such as B, N and BN residues) in the compound.
In one example, the compounds of the present disclosure have the following structure:
(hereinafter referred to as "B", "N", and "BN" residues, respectively), wherein R and R' are independently selected from the group consisting of: straight chain alkaneRadicals (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and the like), branched alkyl radicals (e.g., branched derivatives of propyl, butyl, pentyl, hexyl, heptyl, octyl, and the like), and ether and oligoether radicals (e.g., - (CH)2)2OCH3、-(CH2)2OCH2CH3、-(CH2)2OCH2CH(CH3)2、-(CH2)2O(CH2)2CH(CH3)2Etc.); x is an acyl group (e.g., acetyl, trifluoroacetyl, phenylacetyl, fluorenylmethoxycarbonyl, etc.); y is i) -NHCH3、-NHCH2CH3-NHR "or-NHAr, or ii) -OCH3、-OCH2CH3、-OCH2CH3、-OC(CH3)3OR-OR "where Ar is aryl and R" is a straight OR branched chain alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, etc.); and wherein n is 1 to 64, including all integers and ranges therebetween. In many instances, n is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In many instances, n is 4, 5, or 6.
In one example, a compound of the present disclosure, for each aromatic substituent, at least one substituent (e.g., R and/or R') of the aromatic substituent bonded to an oxygen and hydrogen bonded to an amide hydrogen must be methyl. For example, for each backbone amide bond, at least one substituent (e.g., R and/or R') on the aromatic substituents on either side of the amide bond must be methyl.
In various examples, each substituent (e.g., R and/or R') on an aromatic substituent can be the same, different, or a combination of substituents. In some non-limiting illustrative examples, the aromatic substituent of a compound of the present disclosure has one of the following structures:
in one example, the compounds of the present disclosure form a folded tubular structure (e.g., a helix). In one non-limiting illustrative example, the compound folds as shown in the following structure (using B aromatic substituents as examples, and not excluding N, BN and its combinations (including those with B)):
where starting from the bold end of the structure moving clockwise, the compound spirals down into the page.
The compounds of the present disclosure may form helices (and thus may be referred to as helices or helix compounds). The helix may be right-handed or left-handed.
In one example, the helix comprises a compound having 6.5 residues per turn (e.g., the residues are aromatic substituents having, for example, but not limited to, structure B, N, BN or a combination thereof). The spiral may comprise about one turn per spiral
The spiral of the present disclosure has an inner portion and an outer portion. In one example, the interior of the helix is a hollow tubular cavity containing hydrophilic groups/moieties. In one example, the exterior of the helix comprises hydrophobic groups/moieties.
The interior of the helix has the widest interior linear dimension (e.g., inner diameter). The widest inner linear dimension of the inner part is 3.5 to
In one example, the widest internal linear dimension (e.g., inner diameter) in the compounds of the present disclosure can vary. In such an example, the spiral may include different segments, each segment having a different widest internal linear dimension. For example, a segment of a helix comprising at least one turn may haveThe widest inner linear dimension of (a). In a second section of the helix comprising at least one turn, there may be
In one example, the helix has the longest linear dimension (e.g., length). Longest lengthLinear size of 3.5 to
Desirably, the substituents (e.g., R and/or R') on the aromatic substituents have a moderate hydrophilicity. In one example, the compounds of the present disclosure are soluble in polar aprotic solvents (e.g., N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), etc.). For example, the compounds of the present disclosure maintain solubility at millimolar concentrations (e.g., soluble at concentrations of 0.1 to 10mM, including all 0.1mM values and ranges therebetween).
In one aspect, the present disclosure provides compositions comprising a compound of the present disclosure. In one example, a plurality of compounds of the present disclosure are assembled such that the compounds are stacked on top of each other to form a cylindrical structure. The cylindrical structure is a composite of assembled compounds, and the longitudinal axes of each compound are coaxially aligned. The cylindrical structure has an interior and an exterior. The interior of the cylindrical structure is a continuous hollow tubular cavity. Other assemblies are contemplated and are within the scope of the invention.
The compositions of the present disclosure may form a helix (and thus may be referred to as a helix or a spiral composition). The helix may be right-handed or left-handed.
In one example, the composition comprises a helix having 6.5 residues per turn (e.g., the residues are aromatic substituents having, for example, but not limited to, the structure B, N or BN). The spiral may comprise about one turn per spiralA compound of pitch (c). The pitch and number of residues per turn is determined by the bond angle of the aromatic substituent. Without being bound by any particular theory, these bond angles may vary by a few degrees depending on the temperature. Thus, it is expected that the number of residues and pitch per turn will not be exactly 6.5 residues and
In one example, the interior of the composition comprises hydrophilic groups/moieties. In one example, the outer portion comprises a hydrophobic group/moiety.
The interior of the composition of the present disclosure has the widest linear dimension (e.g., diameter). The widest inner linear dimension of the inner part is 3.5 toIncluding all therebetween
The composition has the longest linear dimension (e.g., length). Longest linear dimension of 3.5 toIncluding all therebetweenRange of values and. In another example, the longest linear dimension is 4 to
In one example, a composition of the disclosure comprises a helical compound of the disclosure, wherein each helical compound has a different longest linear dimension (e.g., length). For example, the composition comprises a longest linear dimension ofOf longest linear dimension ofAnd the longest linear dimension ofThe third compound of (1).
In one example, a composition of the disclosure can comprise a helical compound of the disclosure, wherein each helical compound comprises a different plurality or combination of aromatic substituents. For example, the composition of the present disclosure may contain a first helix compound comprising a BN aromatic substituent, a second helix compound comprising a B aromatic substituent, and a third helix compound comprising an N aromatic substituent. In another example, a composition of the present disclosure may contain a first helix compound comprising a BN aromatic substituent and a B aromatic substituent, a second helix compound comprising a BN aromatic substituent, and a third helix compound comprising an N aromatic substituent.
In one example, the compositions of the present disclosure are soluble in polar aprotic solvents (e.g., N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), etc.). For example, the compositions of the present disclosure maintain solubility at millimolar concentrations (e.g., soluble at concentrations of 0.1 to 10mM in polar aprotic solvents, including all 0.1mM values and ranges therebetween).
In one aspect, the present disclosure provides the use of compounds and/or compositions of the present disclosure. In various examples, one or more compounds and/or compositions of the disclosure are used to form pores in vesicles (e.g., to form transmembrane pores in liposomes and/or cells). The pores may be nanopores. In various examples, the compounds and/or compositions of the present disclosure can be administered to a cell, tissue, organ, or individual (e.g., an individual in need thereof). The subject may be a human, a non-human mammal, a non-mammal, or a plant.
Short spiral folds (e.g., those having one to five spiral turns) may be stacked on top of each other and form a self-assembled pore across a lipid bilayer (e.g., cell membrane), while long spirals (e.g., a single fold having up to ten to twelve spiral turns) may serve as a single-molecule pore across a lipid bilayer. Self-assembled pores, especially those consisting of stacked short helices, are stable at low temperatures, but are broken at high temperatures by thermal motion. Such self-assembled pores can be used to develop thermally responsive (i.e., temperature responsive) methods for delivering target molecules into cells. In one example, the stacked short helices are stable at 1-40 ℃ (including each 0.1 ℃ value and range therebetween). In many instances, the stacked short helices are stable at 1-37 ℃ (including each 0.1 ℃ value and range therebetween).
In various examples, the compounds and/or compositions of the present disclosure are used in cryoprotective methods, nanopore-facilitated transport and/or uptake of therapeutic and/or diagnostic molecules, and/or other pharmaceutical or biological applications. For example, the compounds and/or compositions of the present disclosure may be used in methods of delivering drugs (e.g., therapeutic drugs), nutrients, imaging agents, radioactive or fluorescent tracers, or combinations thereof (e.g., as membrane-bound sensor molecules and ions (e.g., in methods for detecting chemical or biological warfare-like toxic proteins and bacteria (e.g., anthrax)) and as nanopore arrays (membranes) (which may be used, for example, as materials or methods for separating and purifying, for example, molecules and ions).
The compounds and/or compositions of the present disclosure may form transmembrane pores. For example, the membrane is a membrane of a liposome, cell, or other similar vesicle or molecular system. The compounds and/or compositions of the present disclosure may be referred to as pore-forming compounds and/or compositions.
In one example, the compounds and/or compositions of the present disclosure form a single molecule or self-assembled pore having a length that matches the thickness of the lipid bilayer or cell membrane (typically-3.6 to 4.0 nm). For example, compounds that span the entire length of the membrane are referred to as molecular pores and/or single-molecular pores. For example, the composition spanning the entire length of the membrane is a self-assembled pore.
In one example, a method of the present disclosure includes contacting (e.g., at a temperature at or below physiological temperature) a compound and/or composition of the present disclosure (e.g., a compound and/or composition of the present disclosure dissolved in a solvent (e.g., water or a mixture of water and one or more polar aprotic solvents such as DMSO or DMF)) with a vesicle (e.g., a liposome and/or a cell or similar system) having a membrane and thereby causing the compound and/or composition to form a pore across the membrane thickness (e.g., a transmembrane pore).
Target molecules can be transported through pores (e.g., transmembrane pores) formed by the compounds and/or compositions of the present disclosure. In many instances, the target molecule is a hydrophilic species. Thus, in various examples, the methods of the present disclosure include contacting a membrane of a vesicle (e.g., a liposome and/or a cell or similar system) having a transmembrane pore with a target molecule, where the target molecule is transported into the vesicle (e.g., a liposome and/or a cell or similar system). In various additional examples, a molecule of interest (e.g., a hydrophilic species) is transported out of a cell. Non-limiting examples of target molecules include ions (e.g., protons, sodium, potassium, or chloride ions), dyes (e.g., 8-hydroxypyrene-1, 3, 6-trisulfonate trisodium salt (HPTS)), nutrients commonly used in cell or tissue culture, small molecules (e.g., drugs, such as anticancer drugs, 5-fluoro-2' -deoxyuridine (5-FdU), etc.), carbohydrates (e.g., pentoses and hexoses, such as, but not limited to, mannose, glucose, galactose, etc.; disaccharides, such as, but not limited to, sucrose, trehalose, etc.); polyols (also referred to herein as "sugar alcohols," such as mannitol, sorbitol, and the like), chelating agents for metal ions (e.g., hydrophilic chelating agents for metal ions, such as hydrophilic chelating agents for fe (ii) and/or fe (iii)), cryopreservatives (CPAs, such as, but not limited to DMSO, ethylene glycol, and propylene glycol), peptides, and combinations thereof. Examples of suitable CPAs, ions and nutrients are known in the art.
In many instances, compounds containing BN and/or N residues are desirable for transporting a molecule of interest into or out of a cell.
In nanopore-facilitated transport and/or uptake of therapeutic molecules, applications of the technology may include, but are not limited to: intracellular delivery of drugs (e.g., hydrophilic drugs) and/or adjuvants for biomedical research and application in biopharmaceuticals for cancer and other diseases; intracellular delivery of antigens, antisense nucleic acids (DNA and RNA) for gene therapy and the like; and other medical applications such as cell therapy (e.g., chimeric antigen receptor therapy (CAR-T)) and blood therapy.
In exemplary applications, drug delivery and/or drug acquisition assistance includes, but is not limited to, delivery of one or more drugs or agents (e.g., intracellular delivery of hydrophilic drugs and/or adjuvants) to a live organ, a non-live organ, a specimen, bone marrow, blood, stem cells, etc., of an animal (e.g., a mammal). The animal may be a human or non-human. Exemplary drug delivery applications may include related drug formulations, devices, delivery methods, and/or other drug acquisition aids or methods.
The techniques and related methods may also be used for other pharmaceutical or biological applications, such as medical or biomedical applications. Exemplary medical applications may include, but are not limited to, therapies or treatments involving cell therapy (e.g., CAR-T), nucleic acids (DNA/RNA), bone marrow, blood or blood components, gene therapy, and the like. Exemplary biomedical applications may include, but are not limited to, biopharmaceutical research for drugs against cancer and other diseases, cell and bacteria freezing (e.g., escherichia coli and escherichia coli competent cells, staphylococci, etc.).
Non-limiting examples of materials suitable for use in connection with applications involving the compounds and compositions of the present disclosure include mammalian cells (human and non-human), tissues, hybridoma cells, viruses, bacteria, parasites, fungi, and the like. Other suitable materials include agricultural or other plants, tissues and cells.
In one example, the molecules and/or self-assembled pores of the present disclosure are used in a detection method (e.g., as a sensor). The detection method comprises the formation of molecules and/or self-assembled pores in the membrane of vesicles (e.g. liposomes or similar systems), wherein the vesicles optionally encapsulate stimulus-responsive molecules (e.g. fluorescent dyes). Without being bound by any particular theory, a target molecule (e.g., an analyte, non-limiting examples of analytes include carbohydrates, peptides, dyes, and/or ions, etc.) is transported into or out of the vesicle, where the target molecule interacts with or binds to the pore, which interferes with the flow of ions through the pore. Ion transport through the membrane-bound pores is sensed by an encapsulated stimuli-responsive molecule (e.g., a fluorescent dye). The change in the local concentration of the target molecule is measured by a change in the fluorescence emission of the encapsulated stimulus-responsive molecule (e.g., a fluorescent dye).
Methods of using the compounds and/or compositions of the present disclosure include: i) contacting the membrane of a vesicle (e.g., a liposome, a cell, or a similar vesicle or molecular system) with at least one compound and/or composition of the present disclosure such that the compound and/or composition forms pores in the membrane of the vesicle, wherein the vesicle optionally encapsulates a stimulus-responsive molecule (e.g., a fluorescent dye); ii) contacting an analyte (e.g., a solution comprising an analyte, such as a carbohydrate, peptide, dye, and/or ion) with the vesicle such that the analyte interacts (e.g., binds) with the pore; and iii) measuring a change in fluorescence emission of the encapsulated stimulus-responsive molecule.
The compounds and/or compositions of the present disclosure can be used to mediate proton transport (e.g., for detecting the presence or absence or concentration of protons in a sample). For example, an effective amount of an acid or base (e.g., NaOH) that changes the pH of the solution is added to vesicles comprising a solvent and an encapsulated pH sensitive molecule (e.g., a dye, such as HPTS) or drug (e.g., a hydrophilic drug, such as 5-FdU) to form a mixture, and an amount of a second solution comprising a solvent (e.g., DMSO or DMF) and a compound and/or composition at a millimolar concentration (e.g., 0.1mM) is added to the mixture. Proton transport is measured by the change in fluorescence emission intensity of the encapsulated dye over time.
In one aspect, the compounds and/or compositions of the present disclosure may be used for molecular scale chromatography.
In one example, the transmembrane pore of the present disclosure can be used to selectively transport or deliver a molecule of interest (e.g., a carbohydrate, peptide, Cryoprotectant (CPA), dye, and/or ion) into and/or out of a vesicle (e.g., a liposome and/or a cell or similar system) having a membrane.
In various examples, the target molecule used in the method is a Cryoprotectant (CPA). In biological preservation based on cryoprotection, applications of the technique may include, but are not limited to, the following preservation: cells such as e.coli (e.coli) and e.coli competent cells, Staphylococcus (Staphylococcus) and the like, mammalian cells, hybridoma cells, viruses, tissues, parasites and fungi; plant tissues, seeds, specimens, food, fruits and vegetables, etc.; animal tissue, specimens; an organ; meat (food), etc.; and human organs, specimens, blood, stem cells, etc.
Exemplary cryopreservation applications can involve the delivery of CPAs to cells, tissues and organs of humans, non-human animals, plants, and the like. For humans, exemplary cryopreservation applications can include, but are not limited to, delivery of CPAs to living organs, non-living organs, samples, blood (e.g., stem cells), etc., of humans. For non-human animals, exemplary cryopreservation applications may include, but are not limited to, tissue freezing of non-human animal food, specimens, organs, and the like. For plants, exemplary cryopreservation applications may include, but are not limited to, plant tissue, seeds, specimens, food, fruits and vegetables, and the like.
The compounds and/or compositions are expected to prevent ice formation by facilitating the transport of CPAs into cells. It is contemplated that the compounds and/or compositions will form single molecule or self-assembled transmembrane pores that can act as molecular channels to facilitate safe and effective intracellular delivery and removal of target molecules (e.g., CPAs during cryopreservation). These rationally designed synthetic transmembrane pores are expected to serve as selective transmembrane channels to transport target molecules. For example, these rationally designed unimolecular and/or self-assembling transmembrane pores are expected to serve as thermally responsive transmembrane channels to transport CPA at temperatures ≦ 3 ℃ where protein channels typically fail. This disruption of the transmembrane channel at elevated temperatures (e.g. 37 ℃) will prevent the deleterious effects of the non-selective pore on the growing cells. As a result, CPA exposure time of cells reaching ice-free cryopreservation temperature can be significantly reduced. By reducing the formation of intracellular ice and also by eliminating non-selective open channels at temperatures at which cells return to normal growth, cell yield and viability will be greatly enhanced after storage.
It is expected that the compounds and/or compositions of the present disclosure will promote intracellular delivery and transmembrane equilibrium of target molecules (e.g., CPAs). It is contemplated that size and function tunable temperature responsive synthetic transmembrane pores comprising one or more compounds and/or compositions of the present disclosure will serve as efficient molecular channels that remain open at sub-zero temperatures for efficient delivery of target molecules (e.g., CPAs) across cell membranes. In the case where the target molecule is CPA, this will significantly reduce the CPA exposure time and the loading/unloading temperature during freezing. This temperature-responsive characteristic or other engineered environmental sensitivity stimuli will allow the synthesized nanopore to be closed at or above physiological temperatures, which provides minimal interference with membrane integrity and low toxicity. This approach is expected to be effective when CPA is loaded into tissue using a "liquidus tracking" or step-wise approach, where increasing concentrations of CPA solution are loaded into the tissue/organ at progressively lower temperatures.
Cryoprotectants (CPAs) are additives that improve the post-thaw viability of cryo-preserved biological systems from cells to large and complex tissues/organs by preventing ice crystal nucleation and growth. Membrane-permeable CPAs can also prevent osmotic shrinkage of cells and reduce the amount of available water by permeation and equilibration across the cell membrane. All known CPAs exhibit various levels of cytotoxicity at effective concentrations, and this toxicity can be reduced by lowering the CPA loading temperature and exposure time. However, most CPAs are effectively impermeable at temperatures below zero.
In one example, one or more compounds and/or one or more compositions of the present disclosure (which may form a synthetic nanopore comprising one or more spiral folds of the present disclosure) or a composition of the present disclosure is delivered (e.g., administered) into a target system (e.g., a mammalian organ or tissue and mammalian and non-human mammals). Methods of administration are known in the art, and non-limiting examples thereof are described herein.
In one example, one or more compounds and/or one or more compositions of the present disclosure (which may form a synthetic nanopore comprising one or more macrocyclic compounds of the present disclosure) are delivered to a target system (e.g., an organ or tissue (e.g., a mammalian organ or tissue)) at physiological temperature (e.g., 37 ℃) followed by CPA loading at low temperature (e.g., <4 ℃). During cooling, a high influx rate of CPA through the nanopore can be maintained as a function of the concentration gradient across the cell membrane, thereby reducing the time required to reach a vitrification concentration. Upon reheating, the nanopores, particularly the self-assembled pores, will be closed due to increased instability at high temperatures. Thus, at temperatures at or above physiological temperatures, the nanopores will be sealed and will diffuse out of the system, resulting in low toxicity. This use of synthetic nanopores can significantly reduce toxicity and cell damage caused by CPA and salt induced osmotic shrinkage during cooling and reheating by (1) reducing CPA exposure time and (2) enabling rapid CPA loading at lower temperatures.
In addition, by modifying the inner lumen of the spiral folds, versatile functional organic nanotubes with various sizes and properties allow selective CPA transport while preventing ion exchange. The introduction of functional supramolecular assemblies to enhance membrane permeability of CPAs may lead to revolutionary solutions for long-term cryopreservation of large/complex tissues/organs, thereby making possible the realization of "organs on demand".
The compounds and/or compositions of the present disclosure may be provided as pharmaceutical compositions for administration by combining them with any suitable pharmaceutically acceptable carrier, excipient, stabilizer, or combination thereof. Examples of pharmaceutically acceptable carriers, excipients and stabilizers can be found in Remington, The Science and Practice of Pharmacy (2005), 21st edition, Philadelphia, PA.Lippincott Williams & Wilkins. For example, suitable carriers include excipients and stabilizers that are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetic acid, Tris, phosphoric acid, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives, such as octadecyl dimethyl benzyl ammonium chloride; (ii) hexanediamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; tonicity adjusting agents (tonicifiers), such as trehalose and sodium chloride; sugars, such as sucrose, mannitol, trehalose, or sorbitol; surfactants, such as polysorbates; salt-forming counterions, such as sodium; and/or a non-ionic surfactant, such as a Tween (Tween) or polyethylene glycol (PEG). The pharmaceutical composition may comprise other therapeutic agents. The compositions of the present invention may be provided in a single dose or in multiple doses, covering all or part of the treatment regimen. The composition may be provided as a liquid, solid, semi-solid, gel, mist, vapor, or any other form that can deliver it to a subject.
Administration of formulations comprising compounds and/or compositions described herein can be performed using any suitable route of administration known in the art. For example, formulations comprising a compound and/or composition of the present disclosure may be administered by intravenous, intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, oral, topical, or inhalation routes. The composition may be administered parenterally or enterally. The composition may be introduced as a single administration or multiple administrations, or may be introduced in a continuous manner over a period of time. For example, administration may be a predetermined number of administrations or daily, weekly or monthly administration, which may be continuous or intermittent, as may be clinically desirable and/or therapeutically indicated.
The steps of the methods described in the examples disclosed herein are sufficient to perform the methods of the present disclosure. Thus, in one example, a method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.
In the following statements, various examples of the compounds, compositions, and methods of using the compounds and compositions of the present disclosure are described:
wherein the compound has a bent backbone that is stabilized at least in part by rigidifying each amide group with an amide bond of each aromatic substituent by intramolecular hydrogen bonding, and at least in part by interactions between the aromatic substituents,
wherein the composition comprises a plurality of aromatic substituents having the structure:
wherein R and R' are independently at each occurrence selected from the group consisting of: straight chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and the like), branched alkyl groups (e.g., branched derivatives of propyl, butyl, pentyl, hexyl, heptyl, octyl, and the like), and ether and oligoether groups (e.g., - (CH)2)2OCH3、-(CH2)2OCH2CH3、-(CH2)2OCH2CH(CH3)2、-(CH2)2O(CH2)2CH(CH3)2、Etc., wherein the asterisks indicate a stereogenic carbon (i.e., a carbon having a stereochemical R-type or S-type), n is 1, 2, 3, 4, 5, or 6, and R' "is a straight or branched alkyl group (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, isopentyl, etc.), and combinations thereof;
x is an acyl group (e.g., acetyl, trifluoroacetyl, phenylacetyl, fluorenylmethoxycarbonyl, etc.);
y is
i)-NHCH3、-NHCH2CH3-NHR' and-NHAr, or
ii)-OCH3、-OCH2CH3、-OCH2CH3、-OC(CH3)3And OR's,
wherein Ar is aryl and R' is a straight or branched chain alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and the like); and is
n-1 to 64, including all integers and ranges therebetween.
Statement 2. the compound of
Statement 3. the compound of statement 2, wherein the helix is internal and external and the hydrogen bond is external to the helix.
Statement 4. the compound of statement 2 or 3, wherein the helix has about 6.5 residues per turn.
Statement 5. the compound of any of statements 2 to 4, wherein the helix has about each turn
Statement 6. the compound of any of statements 3-5, wherein the interior is a hollow tubular cavity parallel to the longitudinal axis.
Statement 7. the compound according to any one of statements 3 to 6, wherein the inner diameter of the inner portion is from 3.5 toIncluding all therebetweenValues and ranges (e.g. 7 to
Statement 8. the compound of any of statements 2 to 7, wherein the length of the compound (e.g., the length along the longitudinal axis) is from 3.5 to 3.5Including all therebetweenValues and ranges.
Statement 9. the compound of any of statements 3 to 8, wherein the inner portion (also referred to as internal pores) is hydrophilic and the outer portion is hydrophobic.
Statement 10. a spiral composition comprising an assembly of identical compounds according to any one of the preceding statements or an assembly of a mixture of different compounds according to any one of the preceding statements, wherein each of the identical compounds or each of the mixture of different compounds is disposed (i.e., stacked) on an adjacent compound (e.g., with the longitudinal axes of each compound coaxially aligned) to form a cylindrical structure.
Statement 11. the spiral composition of statement 10, wherein the cylindrical structure has an exterior and an interior.
Statement 12. the spiral composition of statement 10 or 11, wherein the interior is a continuous hollow tubular cavity.
Statement 13. the spiral composition of any of statements 10 to 12, wherein the spiral composition has a length (e.g., length along the longitudinal axis) of 3.5 to 3.5
Statement 14. a method of using a compound according to any one of
Statement 15. the method of statement 14, comprising contacting a compound of any one of
Statement 16. the method of any of statements 14 to 15, comprising transporting a target molecule (e.g., a hydrophilic compound and/or a hydrophilic species, such as a carbohydrate, a polyol, a proton, an ion, a dye, a peptide, a CPA, a drug, an adjuvant, or a combination of any of the foregoing) through the pore.
Statement 17. the method of any of statements 14-16, wherein the contacting comprises administering the compound and/or the composition to an individual (e.g., an individual in need of treatment).
Statement 18. the method of statement 17, wherein the method further comprises administering to the subject a molecule of interest (e.g., a hydrophilic compound and/or a hydrophilic substance, such as a carbohydrate, e.g., glucose, sucrose, trehalose, and the like, a polyol, e.g., sorbitol and the like, glycerol, a proton, an ion, a chelator of metal ions (e.g., a hydrophilic chelator of metal ions, such as hydrophilic chelators of fe (ii) and fe (iii)), a dye, a peptide, a CPA, e.g., an anti-freeze peptide, a non-natural anti-freeze oligomer, e.g., a peptoid, combinations thereof, and the like, a drug, an adjuvant, or a combination of any of the foregoing).
Statement 19. the method of any one of statements 14 to 18, wherein the method is performed in vivo, in vitro, or ex vivo.
Statement 20. a composition comprising at least one compound according to
Statement 21. the composition of statement 20, wherein the pore is stable at temperatures of 1 to 40 ℃, including all 0.1 ℃ values and ranges (e.g., 1 to 37 ℃).
Statement 22 the composition of statement 21 or 22, further comprising a plurality of the same or different compounds.
Statement 23. the composition of any one of statements 20 to 22, wherein a plurality of compounds self-assemble into a supramolecular structure.
Statement 24. the composition of any of statements 20 to 23, wherein at least one compound is a helix extending longitudinally in the direction of the longitudinal axis.
Statement 25. the composition of any one of statements 20 to 24, wherein the helix is dextrorotatory or levorotatory.
Statement 26. the composition of any of statements 20-25, wherein the helix has about 6.5 residues per turn.
Statement 27. the composition of any of statements 20 to 26, wherein the helix has about each turnThe pitch of the thread.
Statement 28. the composition of any of statements 20 to 27, wherein the composition has a length along the longitudinal axis of from 3.5 toIncluding all therebetween
Statement 29. the composition of any of statements 20-28, wherein the tubular structure has an interior and an exterior.
Statement 30. the composition of any of statements 20 to 29, wherein the interior is hydrophilic.
Statement 31. the composition of any of statements 20-30, wherein the interior is a hollow tubular cavity parallel to the longitudinal axis.
Statement 32. the composition of any of statements 20 to 31, wherein the inner diameter of the interior is from 3.5 to 15, including all therebetween
Statement 33. the composition of any of statements 20 to 32, wherein the exterior of the helix has one or more hydrogen bonds.
The following examples are presented to illustrate the present disclosure. They are not intended to be limiting in any way.
Example 1
This example provides a description of the oligomeric amide folds and aggregated folds of the present disclosure, methods of making the same, and characterization thereof.
Materials-aromatic oligoamides, in which the backbone amide bonds are constrained (or rigidized) by highly favorable intramolecular hydrogen bonds, are found to fold into pore-containing helical conformations that are stable in a variety of solvents, from non-polar to polar solvents, including aqueous media. The technology involves three classes of aromatic oligoamides consisting of (1) benzene (B series), (2) naphthalene (N series), and (3) benzene and naphthalene (BN series) residues (fig. 1), which fold into conformations with hydrophilic internal pores of different diameters based on the same backbone rigidizing folding mechanism.
Three oligomeric amide series folded in the same way, with a helix having-6.5 residues per turn and
FIG. 2 shows the energy-minimized folded structures of 8-, 16-, 32-and 64-mers of B-series oligoamides. The length of each of these helices is defined by the chain length (i.e., number of residues) of the corresponding oligomer. Each oligomer of the N and BN series follows the same predictable correlation between its chain length (i.e. number of monomer residues) and the length of the folded helix structure as the B series shown in figure 2.
Properties and functions-the transmembrane pore formed by a helix can be a self-assembled pore, i.e. a pore that requires multiple molecules to be stacked on top of each other to span the membrane; or it may be molecular pore, i.e. of sufficient length
With oxygen decorated hydrophilic interiors of varying sizes, these three types of hollow folds act as pores that transport cationic and hydrophilic molecules (e.g., various sugars) across the lipid bilayer (cell membrane). Although the diameter of these hydrophilic pores is large enough to allow passage of substantially all sizes of cations, the different diameters of the three types of pores will be suitable for transporting different molecules. The diameter of the series B pores allows the passage of sugars having 3 to 5 carbons and linear sugar alcohols. Diameter of BN series of holesAnd should be able to transport monosaccharides such as glucose, fructose, mannose, and many non-branched di-and oligosaccharides. N series helix providing
Method-synthesis of aromatic oligoamides. The general procedure for the synthesis of the series B oligoamides is shown in figure 3. The same synthetic procedure is equally applicable to the preparation of N and BN series oligoamides.
Coupling of the monomeric acid chlorides and amines gave dimers (2 mers) which were reduced to dimeric amines (2 mers-NH 2). Protection of the 2mer-NH2 with a Cbz group (and similarly with an Fmoc or TFA group) followed by removal of the t-butyl group affords dimer acid (Cbz-dimer acid). Dimer acid and dimer amine were coupled with HBTU in DMF at 80 degrees celsius to give tetramer (4mer), which was then converted to 4-mer acid and 4-polyamine. The same coupling procedure was repeated to give octamers (8 mers) followed by 16, 32 and 64 mers. This synthesis strategy of doubling the length allows the synthesis of long oligoamides rapidly with a controllable number of coupling steps.
Transmembrane proton transport mediated by B-series oligomeric amide foldings. The ability of the B series 8-mer, 16-mer, 32-mer and 64-mer to mediate proton transport across lipid bilayers was evaluated using a vesicle-based assay. To a solution of Large Unilamellar Vesicles (LUVs) with HPTS (pH sensitive fluorescent dye) encapsulated at pH 7.4, an aliquot of 1N NaOH is added, which increases the pH outside the vesicles, forming a proton gradient across the lipid bilayer. An aliquot of a solution of one of the oligoamides (0.1mM) dissolved in DMSO was added to the LUV solution to a final concentration of 0.5 μ M and the fluorescence emission of the encapsulated HPTS was followed over time. An increase in emission intensity of the encapsulated HPTS indicates a proton transport output LUV. Finally, detergent Triton X-100 was added to rupture all vesicles (capture), releasing TPTS into the bulk solution. The results are shown in FIG. 4.
As expected, all oligomers showed significant to significant activity in proton transport. The longest 64-mer should form a single-molecule transmembrane pore, which shows the highest activity. Proton transport activity can be directly related to the length of the oligoamide, with longer exhibiting higher activity. Similar trends in transporting other cations and small hydrophilic molecules (e.g., glyceraldehyde, glycerol, other linear sugar alcohols such as sorbitol, xylitol, etc.) can also be transported through the pores of the B-series oligoamide foldamers and across the lipid bilayer.
Example 2
This example provides a description of the synthesis of the N series.
The following is an example of the synthesis of N series monomers with methyl groups shown in the following scheme:
while the present disclosure has been described with respect to one or more particular embodiments and/or examples, it will be understood that other embodiments and/or examples of the disclosure may be made without departing from the scope of the disclosure.
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