Supramolecular complexes of biomolecules

Abstract

The present invention provides a ternary complex of a cucurbituril subject, such as CB [8], with a first aryl guest and a second aryl guest, where the first aryl guest is a group within a biomolecule, such as insulin, and the first and second aryl guests are different. Also provided is a method of de-compositing a complex, the method comprising the steps of treating the complex with a competing guest, and allowing the competing guest to replace at least a first aryl guest from the complex.

Description

[0001] The project leading to this application has received funding from the European Union's Horizon 2020 research and innovation programme (European Research Council grant agreement No. 726470 and Marie Sklodowska-Curie grant agreement No. 845640).

[0002] Related Applications

[0003] This application claims priority to and the benefit of GB 2205916.6 filed on April 22, 2022 (22.04.2022) and GB 2206222.8 filed on April 28, 2022 (28.04.2022), the contents of each of which are incorporated herein by reference in their entirety. Technical Field

[0004] The present invention provides a method for capturing and stabilizing biomolecules such as polypeptides using cucurbituril supramolecular complexes. Also provided are complexes and methods for releasing biomolecules from the complexes on demand. Background Art

[0005] Methods for improving the stability and thus the shelf life of biomolecules such as insulin are of interest in the medical field.

[0006] Webber et al. have explored the modification of insulin with poly(ethylene glycol) (PEG) to achieve increased stability of the polypeptide. Here, insulin was incorporated into a cucurbit[7]uril (CB[7])-PEG conjugate. CB[7] is covalently linked to the PEG group. CB[7] forms a binary complex with the phenyl group of the Phe residue at position 1 of insulin. The host-guest complex serves as a substantially permanent link to insulin, wherein the conjugate is used directly in this form in vivo, and the authors suggest that the presence of the CB complex provides advantages for in vivo use (such as possible advantages associated with delayed and controlled uptake of higher molecular weight insulin).

[0007] Webber et al. demonstrated that cucurbit[7]uril can be used to form complexes with other biomolecules, including glucagon and anti-CD20 antibodies.

[0008] However, modifying biomolecules is not always beneficial, and modifications may not always maintain or improve biological activity.A stabilization strategy is needed that allows for the recovery and use of biomolecules in their original form. Summary of the invention

[0009] In a general aspect, the present invention provides a ternary complex comprising a cucurbituril host housing a first aryl guest and a second aryl guest, wherein the first guest is a group of biomolecules, such as a polypeptide.

[0010] The second guest is different from the first guest. Thus, the complex is a heterocomplex. The formation of a heterocomplex may be more favorable than the formation of a homocomplex in which the guests are the same.

[0011] This type of complex can be beneficially used to stabilize biomolecules for storage, and when needed, the biomolecule can be easily and quickly released from the complex. The complex is formed so as to limit the degradation of the biomolecule and maintain its activity when released.

[0012] Selective capture of the biomolecule in the complex also allows separation of the biomolecule from other components in the mixture, thereby also allowing at least partial purification of the biomolecule. For example, the complex can be attached to a solid phase, thereby allowing collection of the complex by simple filtration, separated from other dissolved components of the mixture.

[0013] The first aryl guest may be a carboaryl or heteroaryl group present in the side chain of an amino acid, such as an α-amino acid.

[0014] The first aromatic guest may be a phenyl group, such as present in a phenylalanine residue of a polypeptide. The amino acid residue may be a terminal or mid-chain residue within a polypeptide, and is preferably a terminal residue, such as an amino acid residue within the N-terminal region of a polypeptide, such as an N-terminal residue.

[0015] The polypeptide may be a protein, including an antibody. The protein is preferably insulin, such as human insulin, and includes human recombinant insulin. The first guest may be the phenyl group of the phenylalanine residue at position 1 (1-Phe) of insulin, for example within chain B.

[0016] The second guest can be a perfluorophenyl group (ie, a pentafluorophenyl group). The perfluorophenyl group can be covalently attached directly or indirectly to a solid phase, such as a polymeric solid phase.

[0017] In another aspect of the invention, there is provided a method of forming a complex such as the complex of the first aspect of the invention, the method comprising the steps of mixing a cucurbituril with a biomolecule having a first aromatic guest and a second aromatic guest and allowing the host and guest to self-assemble.

[0018] The complex may be formed by self-assembly. The method may be, and preferably is, carried out under aqueous conditions. The method may be, and preferably is, carried out at ambient temperature, such as at a temperature in the range of 5 to 30°C.

[0019] In another aspect, a method of decomplexing a complex of the present invention is provided, the method comprising the steps of treating the complex with a competing guest and allowing the competing guest to displace at least a first aryl guest, optionally together with a second aryl guest, from the complex.

[0020] The method may be and is preferably carried out under aqueous conditions.The method may be and is preferably carried out at ambient temperature, such as at a temperature in the range of 5 to 30°C.

[0021] The decomplexing process results in the release of the biomolecule from the complex.

[0022] The competing guest may be or contain a compound of a competing guest.

[0023] In a preferred embodiment, the competing compound is memantine (DMADA), which can be used as memantine hydrochloride.

[0024] The release of the biomolecule from the complex may include preliminary steps to form the complex, such as according to the method of the present invention. The release of the complex may be performed 1 week or more after formation, such as 1 month after formation. The complex is stable and prevents degradation of the biomolecule. Therefore, the complex may be suitable for long-term storage of biomolecules, which can be released from the complex when needed without significant loss of biological activity.

[0025] These and other aspects and embodiments of the invention are described in detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shown are: a) host-enhanced isopeptide dimers formed by F'GG, WGG and CB[8]; b) molecular structures of FGG, YGG, and NpGG; c) schematic diagram of on-resin recognition via interfacial heterodimerization.

[0027] Figure 2 a) The WGG (16.0 mM) titration into F'GG-CB[8] (1.0 mM) is shown. 1 H and 19 F NMR spectrum (D2O, 298K); b) HR ESI-MS spectrum of the isopeptide dimer of 1:1 complex of WGG, FGG, YGG, NpGG with F'GG-CB[8] (H2O, 1.0 mM); c) F'GG (3.0 mM) into CB[8] (0.1 mM); d) ITC titration plot of WGG, FGG, YGG, NpGG (3.0 mM) into F'GG-CB[8] (0.2 mM) (10 mM phosphate buffer, pH 7.0, 298K); and e) Schematic diagram of the self-sorting mechanism.

[0028] Figure 3Shown are a) the molecular structure of WGGGGG-dansyl, and b) a schematic diagram of the interface recognition with F'GGGGG resin (10 mM); confocal fluorescence images of c) F'GGGGG resin, and F'GGGGG resin added with d) WGG-CB[8](10 mM), e) WGGGGG-dansyl (10 mM), and F) WGGGGG-dansyl-CB[8](10 mM) obtained by laser excitation at =405 nm under gray field. Scale bar = 200 m. Fluorescence intensity was quantified by mean gray value (MGV).

[0029] Figure 4 Shown are a) a schematic diagram of on-resin identification and separation of aromatic tripeptides from a peptide mixture; b) UV spectra of WGG-CB[8] (1.0 mM) before and after treatment with F'GGGGG-resin (10.0 mM); c) a histogram of the recovery efficiency of WGG in consecutive resin identification cycles; d) the percentage of WGG obtained after multiple cycles of separation; e) a schematic diagram of resin stabilization, on-demand release and binding to CB[8] of insulin, not drawn to scale; f) UV spectra of insulin (0.2 mM) before and after treatment with F'GGGGG-CB[8]-resin (10.0 mM) and then released on days 0, 2, 5 and 8; g) a histogram of the recovery efficiency of insulin on days 0, 2, 5 and 8.

[0030] Figure 5 UV spectra of control experiments for recognition of insulin (0.2 mM) on resin are shown. a) Recognition of insulin (0.2 mM) on F'GGGGG-resin (10 mM); b) Recognition of insulin (0.2 mM) and CB[8] (1.0 mM) on GGGGGG resin (10 mM); c) Recognition of insulin (0.2 mM) on GGGGGG resin (10 mM); d) Recognition of insulin (0.2 mM) and CB[8] (1.0 mM) on blank resin (H-Rink amide ChemMatrix resin) (phosphate buffer, H2O).

[0031] Figure 6 Shown are UV spectra of insulin (0.2 mM) solutions before and after treatment with F'GGGGG-CB[8]-resin (10.0 mM) followed by time-dependent release at 0, 2, 5 and 8 days (phosphate buffer, H2O).

[0032] Figure 7 CD spectra of insulin solutions before and after treatment with F'GGGGG-CB[8]-resin followed by time-dependent release at 2, 5 and 8 days (phosphate buffer, H2O) are shown.

[0033] Figure 8 The selective separation of aromatic peptides on the resin from a mixed peptide mixture (WGG, KGG, EGG, LGG 1.0 mM, phosphate buffer, D2O) in 3 cycles is shown. 1 H NMR. DETAILED DESCRIPTION

[0034] The present inventors have discovered that CB[8] hosts can be used to form complexes with biomolecules (such as polypeptides) and a second component selected to form heteropairs with a set of biomolecules within the host cavity.

[0035] The complex has been found to stabilize biomolecules such as insulin for storage and release on demand without loss of biomolecule integrity, including structural and functional integrity.

[0036] Li et al. described the use of CB[7] to form binary complexes with insulin or human growth hormone (hGH). Here, CB[7] was covalently attached to a solid phase. Insulin and HGA were released from the complexes after treatment with a competing guest. However, Li et al. did not demonstrate or suggest that the formation of the complex was related to the stabilization of the insulin or hGH biomolecules.

[0037] Also, the preparation of CB[7] and its covalent attachment to the solid phase is not simple but complex. Therefore, accessibility and scalability are an issue. In contrast, the use of readily available and easily prepared CB[8] and related larger cucurbituril hosts, as described in this case, avoids the problems associated with the use of CB[7] and its derivatives.

[0038] Compound

[0039] The complex of the present invention comprises a cucurbituril host, such as CB[8], in whose cavity a first guest and a second guest are non-covalently accommodated. This is a supramolecular complex and also a heterogeneous complex, and the first guest and the second guest are different.

[0040] Cucurbituril forms a non-covalent bond with each guest.The inventors have previously determined that complexes of cucurbituril are readily formed and can be used to provide strong non-covalent linkages between compounds.

[0041] Complexes can be formed between cucurbiturils and first guests from biomolecules, and the formation of complexes allows for many functional groups within the monomer.

[0042] The complex further comprises a second guest and in a preferred embodiment of the invention the second guest is attached to the solid phase. The formation of the complex also tolerates such solid phase functional groups.

[0043] When the complex contains two guests in the cucurbituril cavity, the association constant K a is at least 10 3 M -2 , at least 10 4 M -2 , at least 10 5 M -2 , at least 10 6 M -2 , at least 10 7 M -2 , at least 10 8 M -2 , at least 10 9 M -2 , at least 10 10 M -2 , at least 10 11 M -2 or at least 10 12 M -2 Preferably, the association constant is at least 10 10 M -2 .

[0044] Cucurbituril accommodates two guest molecules, and the guest molecules are different. The formation of ternary host-guest complex is believed to proceed through an intermediate binary complex.

[0045] Reference to a first guest and a second guest in this context is not a description of the order of the guest complexes together with the cucurbituril host. Thus, an intermediate binary may be formed with either the second guest and the host, or the first guest and the host.

[0046] The formation of the complex is reversible. The separation of the guest from the cucurbituril host is called decomplexation.

[0047] Complex decomplexation to separate host and guest may occur in response to external stimuli, including, for example, competing guest compounds, light, oxidants or reductants, electrochemical potential and temperature changes, etc. In the methods of the present invention, complex decomplexation is performed under conditions that do not damage or disturb the structure of the biomolecule. For this reason, displacement methods involving benign competing guest compounds are advantageous.

[0048] The competitive guest for decomplexing the CB[8]-based network is or comprises amantadine (ADA) or memantine (DMADA). The competitive guest can be used in excess (molar amount) of the guest present in the complex. In one embodiment, the competitive guest has a higher association constant than the guest of the complex.

[0049] The decomplexation reaction is reversible and thus, if desired, can be allowed to reform after removal of the external stimulus, including, for example, removal of the competing guest compound.

[0050] Cucurbituril and CB[8]

[0051] The complex of the present invention has a cucurbituril host. Cucurbituril is a cucurbituril capable of accommodating two guests. Thus, the host can be a CB[8] host, including variants and derivative forms.

[0052] Cucurbiturils capable of forming ternary and binary complexes can be used in the present invention. There are many examples of cucurbiturils useful for forming such complexes in the art.

[0053] Recent studies have demonstrated that cucurbituril compounds have high in vitro and in vivo biocompatibility and extremely low toxicity (see Uzunova et al. Org. Biomol. Chem. 2010, 8, 2037-2042). Therefore, when used with a non-toxic polymer component, the hydrogel of the present invention is also suitable for use in biological systems.

[0054] Cucurbiturils should be able to form ternary complexes. For example, CB[8] is able to form ternary complexes. The same is true for CB

[10] and CB

[12] compounds.

[0055] The cucurbituril may be a CB[8], CB

[10] or CB

[12] compound.

[0056] In one embodiment, the cucurbituril is a CB[8] compound.

[0057] Reference to a cucurbituril compound is a reference to modifications and derivatives thereof.

[0058] Variants of CB[8] may include structures having one or more repeating units that are structurally similar to glycoluril. The repeating units may include ethylurea units. When all units are ethylurea units, the variant is a hemicucurulin. The variant may be a hemicucurulin

[12] uril (as shown below, see also Lagona et al. Angew. Chem. Int. Ed. 2005, 44, 4844).

[0059]

[0060] Cucurbituril derivatives are provided and can be used in the methods described herein. Cucurbituril derivatives are structures having one, two, three, four or more substituted glycoluril units. Substituted cucurbituril compounds can be represented by the following structure:

[0061]

[0062] in:

[0063] n is an integer of at least 8;

[0064] And for each glycoluril unit:

[0065] Each X is O, S or NR 3 ,as well as

[0066] -R 1 and -R 2 are each independently selected from -H and the following optionally substituted groups: -R 3 、-OH、-OR 3 , -COOH, -COOR 3 、-NH2、-NHR 3 and -N(R 3 )2, where -R 3 Independently selected from C 1-20 Alkyl, C 6-20 Carboaryl and C 5-20 Heteroaryl, or wherein -R 1 and / or -R 2 Yes-N(R 3 )2, two-R 3 Together they form C 5-7 Heterocyclic; or -R 1 and -R 2 Together with the uracil framework, it forms C 6-8 Carbon ring C 4-6 Alkylene.

[0067] In one embodiment, one of the glycoluril units is a substituted glycoluril unit. Thus, for n-1 glycoluril units, -R 1 and -R 2 Each is independently -H.

[0068] In one embodiment, n is 8, 9, 10, 11 or 12.

[0069] In one embodiment, n is 8, 10 or 12.

[0070] In one embodiment, n is 8.

[0071] In one embodiment, each X is O.

[0072] In one embodiment, each X is S.

[0073] In one embodiment, R 1 and R 2 Each independently is H.

[0074] In one embodiment, for each unit, R1 and R 2 One of them is H, and the other is independently selected from -H and the following optionally substituted groups: -R 3 、-OH、-OR 3 , -COOH, -COOR 3 、-NH2、-NHR 3 and -N(R 3 )2. In one embodiment, for one glycoluril unit, R 1 and R 2 One of them is H, and the other is independently selected from -H and the following optionally substituted groups: -R 3 、-OH、-OR 3 , -COOH, -COOR 3 、-NH2、-NHR 3 and -N(R 3 )2. In this embodiment, the remaining glycoluril units are as follows: R 1 and R 2 Each independently is H.

[0075] Preferably, -R 3 It is C 1-20 Alkyl, more preferably C 1-6 Alkyl. C 1-20 The alkyl group may be linear and / or saturated. 3 may be independently unsubstituted or substituted. Preferred substituents are selected from: -R 4 、-OH、-OR 4 , -SH, -SR 4 , -COOH, -COOR 4 、-NH2、-NHR 4 and -N(R 4 )2, where -R 4 Selected from C 1-20 Alkyl, C 6-20 Carboaryl and C 5-20 The substituents may be independently selected from -COOH and -COOR 4 .

[0076] In some embodiments, -R 4 with -R 3 In some embodiments, -R 4 Preferably it is unsubstituted.

[0077] When -R 1 and / or -R 2 Yes-OR 3 、-NHR 3 or -N(R 3)2, then -R 3 Preferably C 1-6 In some embodiments, -R 3 Substituted group -OR 4 、-NHR 4 or -N(R 4 )2 is replaced. Each -R 4 All C 1-6 Alkyl, and itself is preferably substituted.

[0078] Cucurbituril guest

[0079] As mentioned above, guest is a compound that can form a host-guest complex with cucurbituril. Therefore, the term complex refers to the establishment of a host-guest complex. The host-guest complex is a ternary complex that comprises a cucurbituril main body and a first guest and a second guest. The first guest is provided as a group on a biomolecule. The second guest is provided as a group on a second component, which is optionally a solid phase.

[0080] Typically, such complexes are based on CB[8] and its variants and derivatives. The first guest and the second guest are not identical.

[0081] In principle, any compound with suitable binding affinity (such as those mentioned in the above complex discussion) can be used in the present invention. The compound used can be selected based on the size of the moieties that are thought to interact with the cavity of the cucurbituril. The size of these moieties may be large enough to allow complexing with only the larger cucurbituril forms.

[0082] In the case of the present invention, the first guest and the second guest are each an aryl group.

[0083] The aryl group may be a carboaryl group or a heteroaryl group, and is preferably a carboaryl group.

[0084] The aryl group may be a monocyclic, bicyclic or tricyclic aryl group.

[0085] The aryl group may be a monocyclic or a bicyclic aryl group.

[0086] An aryl group can be unsubstituted or substituted, such as monosubstituted.

[0087] Examples of carboaryl groups include phenyl, naphthyl and anthracenyl.

[0088] Examples of heteroaryl groups include indolyl.

[0089] The first guest and the second guest are not identical. In some embodiments, the aryl rings can be identical, but the guests differ in the number and / or identity of the substituents of the rings. For example, in a working example of this situation, a phenyl first guest is used with a second guest having a benzene ring, but this ring is perfluorinated.

[0090] Each aryl substituent may be selected from the group consisting of alkyl, halogen, -OH, -OR 1 、-NH2、-NHR 1 、-NR 1 R 2 , where each -R 1 and -R 2 are independently alkyl.

[0091] Alternatively, each aryl substituent may be selected from the group consisting of alkyl, halogen, -OH, -OR 1 、-CN、-NO2、-NH2、-NHR 1 、-NR 1 R 2 , where each -R 1 and -R 2 are independently alkyl.

[0092] The alkyl group can be C 1-6 Alkyl groups, such as C 1-4 Alkyl, such as methyl or ethyl, such as methyl.

[0093] The halogen group may be selected from -F, -Cl, -Br and -I, and is preferably -F.

[0094] Typically, one aryl guest is halogenated, such as fluorinated, and the other aryl guest is not. Preferably, the second aryl guest is halogenated, such as fluorinated. Here, it is also preferred that the first aryl guest is not halogenated, for example not fluorinated.

[0095] The present inventors have discovered that electron-deficient guests, such as halogenated aryl guests, such as perfluorophenyl, favorably form heterocomplexes with electron-rich guests, such as substituted aryl guests.

[0096] The first guest is typically an unsubstituted or monosubstituted aryl group.

[0097] The second guest is typically a substituted aryl group.

[0098] The first guest may be selected from the group consisting of phenyl, naphthyl such as naphthalen-2-yl, hydroxyphenyl such as 4-hydroxyphenyl, and indolyl such as indol-3-yl.

[0099] The first aryl guest may be covalently linked to the methylene group.

[0100] The first guest may be a phenyl group or an indolyl group, including indol-3-yl.

[0101] Preferably, the first guest is a phenyl group.

[0102] In another embodiment, the first guest is an indolyl group, including indol-3-yl.

[0103] The first guest may be present within the side chain of an amino acid residue.

[0104] When the first guest is a phenyl group, the amino acid residue may be phenylalanine.

[0105] When the first guest is a hydroxyphenyl group, an exemplary amino acid residue in which the guest is a 4-hydroxyphenyl group is tyrosine.

[0106] When the first guest is naphthyl, an exemplary amino acid residue in which the guest is naphth-2-yl is (2-naphthyl)alanine.

[0107] When the first guest is an indolyl group, an exemplary amino acid residue in which the guest is an indol-3-yl group is tryptophan.

[0108] The first aromatic guest may be provided within the side chain of a phenylalanine, tyrosine, indole or naphthylalanine residue.

[0109] The second aryl guest can be selected from substituted phenyl, indolyl such as indol-3-yl, naphthyl such as naphthalene-2-yl and anthracenyl such as anthracene-2-yl. Here, the aryl is substituted with one or more substituents. The aryl can be fully substituted. That is, each available ring position can be substituted. Here, each substituent can be halogen, and preferably each substituent is fluorine.

[0110] The second aryl guest may be covalently attached to the methylene group.

[0111] The second aryl guest may be a phenyl group having five halogen substituents, such as five fluorine substituents. Thus, the second aryl guest may be a perfluorophenyl group (pentafluorophenyl group), and it is preferably attached to a methylene group.

[0112] The second aryl guest may be an indolyl group, such as indol-3-yl, having 1 to 5 halogen substituents, such as 1 to 5 fluorine substituents.

[0113] The second aryl guest may be naphthyl, such as naphthalen-2-yl, having 1 to 7 halogen substituents, such as 1 to 7 fluorine substituents.

[0114] The second aryl guest may be an anthracenyl group, such as anthracene-2-yl, having from 1 to 9 halogen substituents, such as from 1 to 9 fluorine substituents.

[0115] The second aryl guest may be present in the side chain of the amino acid residue. When the second guest is a perfluorophenyl group, the amino acid residue may be perfluorophenylalanine.

[0116] The amino acid residue containing the second aromatic guest may be provided within a polypeptide sequence having two or more consecutive amino acid residues, such as three or more, such as 4 or more, such as 5 or more, such as 6 or more amino acid residues.

[0117] The amino acid residue with the second aryl guest can be provided at the N-terminus of the polypeptide sequence. The terminal amino acid residue can be connected to a polypeptide sequence having one or more, such as three or more, such as 4 or more, such as 5 or more amino acid residues that are consecutive to the terminal amino acid residue.

[0118] Preferably, the polypeptide sequence has an amino acid residue with a second aromatic guest at the N-terminus and which is linked to one or more, such as three or more, such as 4 or more, such as 5 or more glycine residues consecutive to the terminal amino acid residue.

[0119] The second aryl guest can be covalently attached to the solid phase directly or indirectly.

[0120] The solid phase can be a polymer phase, such as a polystyrene resin or a poly(ethylene glycol) (PEG) resin, such as Rink amide ChemMatrix resin. Here, the formation of the complex links the biomolecule to the solid phase via non-covalent interactions in the complex.

[0121] Once the biomolecule is immobilized, it can be separated from other non-insoluble components by simple filtration. Subsequent release of the biomolecule from the complex, for example using the decomplexing methods described herein, then provides the biomolecule in a more purified form.

[0122] The aryl guest may be attached to the optionally substituted imidazolium group, such as via a methylene group.The aryl guest may be attached to the 1 position of the imidazolium ring nitrogen, such as via a methylene group.

[0123] The 2-, 4- and 5-positions of the imidazolium group are preferably unsubstituted. The 3-position of the imidazolium group is optionally substituted and may be substituted.

[0124] The imidazolium 3-position may be substituted by an alkyl group (such as C 1-4 alkyl, such as methyl, or ethyl, such as methyl).

[0125] The imidazolium 3-position may be linked to a methylene group.

[0126] The imidazole group may be covalently linked to the solid phase directly or indirectly. Linkage to the solid phase may be via the imidazolium 3-position, such as via a methylene group linked to the imidazolium 3-position.

[0127] An amino acid residue as described herein may be an α-amino acid residue, a β-amino acid residue or a γ-amino acid residue.

[0128] An amino acid residue as described herein may be an α-amino acid residue.

[0129] The amino acid residue may be in the L-form or the D-form, and is usually in the L-form.

[0130] In a preferred embodiment, the first guest and the second guest are both groups within the side chains of amino acid residues. Preferably, each amino acid residue has the same stereoform, such as both amino acid residues have the L-form.

[0131] Biomolecules

[0132] The biomolecule may be selected from the group consisting of polypeptides, polynucleotides and polysaccharides.

[0133] The present inventors have found that the ternary complex of the present invention stabilizes biomolecules such as insulin, thereby preventing its degradation. The complex can be stored without significant loss of structural or functional activity.

[0134] Biomolecules may inherently contain groups suitable for use as guests in the ternary complexes of the present invention. For example, a polypeptide may contain a phenylalanine residue, and the phenyl group in the side chain of this residue may be a suitable guest.

[0135] In other embodiments, a biomolecule can be modified to include a guest aromatic group such that the biomolecule can be retained in the complex.

[0136] The guest group present in the biomolecule can be used to interact with the cucurbituril host. Therefore, the first aryl guest can be provided at the end of the biomolecule, where it is more likely to be freely available for interaction with the host. However, if the guest is available for interaction, the guest group can also be provided with the mid-chain. Preferably, the first aryl guest is provided at the terminal region of the biomolecule.

[0137] In one embodiment, the biomolecule has a molecular weight of at least 100, at least 200, at least 300, at least 1,000 (1k), at least 5,000 (5k), at least 10,000 (10k), at least 15,000 (15k), at least 20,000 (20k), at least 50,000 (50k), at least 100,000 (100k), or at least 200,000 (200k).

[0138] The polypeptide may be a protein, such as a peptide hormone or an antibody.

[0139] Thus, the biomolecule may be selected from the group of polypeptides consisting of:

[0140] Blood factors, such as Factor VIII and Factor IX;

[0141] Thrombolytic agents, such as tissue plasminogen activator;

[0142] Peptide hormones, such as insulin, glucagon, growth hormone, gonadotropin;

[0143] Hematopoietic growth factors, such as erythropoietin and colony-stimulating factor;

[0144] Interferons, such as interferon-α, interferon-β, interferon-γ;

[0145] Interleukin-based products, such as interleukin-2;

[0146] vaccines, such as hepatitis B surface antigen;

[0147] Antibodies, including monoclonal antibodies; and

[0148] Other proteins such as tumor necrosis factor and therapeutic enzymes.

[0149] Preferably, the biomolecule is a peptide hormone, such as insulin. Polypeptides include proteins with post-translational modifications. Polypeptides may also contain non-natural amino acid residues, and polypeptides may also contain amino acid residues modified during post-translational modification.

[0150] The polypeptide may have at least 10 contiguous amino acid residues, such as at least 20, at least 50, at least 100 or at least 500 amino acid residues.

[0151] The working examples in this case use insulin as an exemplary polypeptide and form a connection with a 1-Phe residue in insulin within chain B. Webber et al. have also formed cucurbituril complexes with 1-Phe residues in insulin, although using CB[7] and only binary complexes. Webber et al. have also demonstrated that cucurbituril complexes with other biomolecules (such as other polypeptides, including glucagon and anti-CD20 antibodies) are possible. The skilled person will appreciate that the present invention can be used to form complexes with biomolecules other than insulin.

[0152] The first guest may be provided at the N-terminal region or C-terminal region of the polypeptide, and preferably the N-terminal. Here, the terminal region may be considered as three consecutive amino acid residues at the N-terminal or C-terminal. Typically, the first guest is present in the amino acid residue itself at the N-terminal or C-terminal, and preferably the N-terminal. The inventors have found that when the first guest is present together with the amino acid residue itself at the terminal, the association constant of the first guest may be the largest, and as the first guest is provided at a greater distance from the N-terminal, such as within the second and third amino acid residues from the N-terminal, the association constant decreases.

[0153] Wherein the biomolecule is a polypeptide, the first guest is typically present in an arylalanine residue within the polypeptide.

[0154] Wherein the biomolecule is a polypeptide, the first guest is usually present in a phenylalanine, tyrosine, indole or naphthylalanine residue in the polypeptide.

[0155] The polynucleotide may be DNA, RNA, or related forms such as PNA, and may be single-stranded.

[0156] A polynucleotide may have at least 10 contiguous nucleotides, such as at least 50, at least 100, or at least 500 nucleotides.

[0157] The first guest may be provided at the 3' or 5' terminal region of the polynucleotide.

[0158] The polysaccharide may contain two or more, such as three or more, consecutive sugar groups.

[0159] The biomolecule may be a naturally occurring biomolecule, or may be a variant, such as a mutant, of such a biomolecule.

[0160] The biomolecule may also be in a modified form, for example containing a label for detection or capture. Typically, such functional groups are provided remote from the first guest so as not to interfere with the formation of the complex.

[0161] The biomolecules may also be in a protected form, such as a chemically protected form.

[0162] Typically, the biomolecule itself is not immobilized on the solid phase. Instead, the formation of the complex can be used to non-covalently link the biomolecule to the solid phase through its interaction with a second guest in the complex, wherein the second guest is linked to the solid phase, as shown in the preferred embodiment of the present invention.

[0163] The biomolecule may be provided in an aqueous mixture, and the biomolecule may be provided together with other components. Here, the formation of a complex that preferentially contains the biomolecule over the other components may serve as a process for purifying the biomolecule from these components.

[0164] These other components may not have suitable guest groups for complex formation, or functional groups may be present but unavailable for formation (eg, because the guest is internalized within the other components).

[0165] insulin

[0166] The present invention is well suited for forming complexes where the biomolecule is insulin.

[0167] The insulin may be a polypeptide having an amino acid sequence corresponding to human insulin.

[0168] The insulin may be recombinantly produced insulin. Thus, the insulin may be human recombinant insulin.

[0169] In a preferred embodiment, the first guest is the phenyl group present in the phenylalanine residue present at position 1 of insulin, particularly human insulin.

[0170] Insulin need not be provided in a sustainable purified form. The method of the present invention allows for preferential formation of complexes with insulin over other compounds present. Once formed, the complex can be easily purified from other components, such as by simple filtration, wherein the complex comprises a solid phase-bound component, such as a second compound.

[0171] Complex formation and decomplexation

[0172] The present invention provides a method for preparing the composite of the present invention, which comprises the steps of mixing a cucurbituril host with a biomolecule having a first aromatic guest and a second aromatic guest, and allowing the host and the guest to self-assemble.

[0173] The method may also include the step of at least partially purifying the complex. For example, the complex may be at least partially separated from one or more other components that are not in the complex.

[0174] Preferably, the second aryl guest is bound to the solid phase and thus the resulting complex is also bound to the solid phase.Here, the complex may be at least partially purified by collecting the solid phase, for example by filtering, to separate the complex from other components.

[0175] The present invention also provides a method of decomplexing a complex of the present invention, the method comprising the steps of treating the complex with an external stimulus and allowing the external stimulus to displace at least a first aromatic guest from the complex.

[0176] The release of biomolecules can be achieved as desired and can be considered as release on demand.

[0177] The external stimulus may be the addition of a competing object. The competing object may be or include amantadine (ADA) or memantine (DMADA).

[0178] The formation and decomplexation can be carried out under aqueous conditions.

[0179] Riskily, the forming and decomplexing steps may also be performed at ambient temperature, such as at a temperature in the range of 5 to 30°C.

[0180] Complexation and decomplexation can be performed at temperatures that are not associated with biomolecule degradation, and the integrity of the biomolecule can be preserved during the complexation and decomplexation process.

[0181] Stabilization of biomolecules

[0182] The complexes of the present invention can be used to stabilize biomolecules for storage, and when needed, the biomolecules can be easily and quickly released from the complexes. Thus, complex formation limits the degradation of the biomolecules and maintains their activity when released.

[0183] Here, the complex of the invention is preferably bound to a solid phase. For example, the second guest is attached to a solid phase, such as a polymer phase.

[0184] Thus, the use of the complex to stabilise biomolecules such as proteins is provided.

[0185] The stability of the biomolecules can be assessed by structural analysis, such as circular dichroism, and may include comparison of the secondary and tertiary structure of the biomolecules before and after complexation. The satiability of the biomolecules can be assessed by suitable activity assays, which may include cell-based studies.

[0186] Purification of biomolecules

[0187] Selective capture of the biomolecule in the complex also allows separation of the biomolecule from other components in the mixture, thereby also allowing at least partial purification of the biomolecule. For example, the complex can be attached to a solid phase, thereby allowing collection of the complex by simple filtration, separated from other dissolved components of the mixture.

[0188] Thus, a method for at least partially purifying a biomolecule from a mixture is provided, the method comprising the steps of providing the biomolecule in a mixture (such as an aqueous mixture), wherein the biomolecule has a first aromatic guest; providing a cucurbituril host and a second aromatic guest in the mixture; allowing the host and guest to self-assemble; and subsequently separating the resulting complex from the mixture.

[0189] experiment

[0190] Cucurbit[n]uril (CB[n]) macrocyclic hosts are ideal for binding peptides due to their high binding affinity and diverse guest tropism. 6-11 Urbach and colleagues reported homopeptide dimers between two FGG tripeptides and CB[8], which showed high binding strength (K about 10 11 M -2 ). 6 This homodimer has been used as a supramolecular oligomer, 12 polymer, 13,14 Hydrogel, 15,16 and protein / peptide assembly 17 Versatile building blocks in the design and manufacture of 17 Despite significant progress, a major challenge remains to quantitatively demonstrate the formation of isopeptide dimers without homodimerization.

[0191] Here, we employed CB[8] to mediate heterodimerization of conventional aromatic peptides and unconventional L-perfluorophenylalanine (F')-containing peptides. Figure 1 Recently, we reported that an electron-poor perfluorophenyl first guest and an electron-rich phenyl second guest can exclusively form a CB[8]-mediated heteroternary complex via host-enhanced polar-π interactions. 18,19 Therefore, we hypothesized that the F'-containing peptide (F'GG) would form a 1:1 complex exclusively with CB[8], avoiding homodimerization due to electrostatic repulsion in the 2:1 complex. Subsequent association of various aromatic peptides (e.g., WGG) with the F'GG-CB[8] complex could yield new host-enhanced isopeptide dimers with superior binding strength, Figure 1 a.

[0192] Five model aromatic tripeptides containing L-perfluorophenylalanine 20 (F'GG), L-tryptophan (WGG), L-phenylalanine (FGG), L-tyrosine (YGG), and L-(2-naphthyl)alanine (NpGG) at the N-terminus were designed and prepared. Figure 1 ab. An equimolar mixture of F'GG, CB[8], and XGG should produce exclusively heterodimers rather than homodimers, which are contained in an equilibrium mixture. Two additional series of tripeptides (GGX, GXG) were synthesized containing aromatic amino acids at the C-terminus or mid-chain to study the effects of a range of second guests and their position in the oligopeptide (as shown below). After elucidating the binding thermodynamics, we applied this heterodimerization to achieve on-resin recognition and separation from a mixture of peptides showing high efficiency and selectivity. 21,22 Aromatic tripeptide, Figure 1 c.

[0193]

[0194]

[0195] conduct 1 H and 19 F NMR titration to probe isopeptide dimerization within CB[8], Figure 2 a (and additional data not shown). Titration of WGG into a 1:1 mixture of F'GG-CB[8] resulted in the gradual appearance of indole protons at 6.25-7.10 ppm. These proton peaks exhibited an upfield shift compared to free WGG due to shielding by the CB[8] cavity, indicating that the indole group of F'GG-CB[8]-WGG is located in a different chemical environment than unbound WGG. This titration was also performed by 19 F NMR monitoring, Figure 2a; A new set of fluorine peaks gradually appeared, while the peak of F'GG-CB[8] disappeared. In addition, the equivalent mixture of F'GG, CB[8] and XGG was characterized by high-resolution ESI-MS. Figure 2 b. All ion peaks of the F'GG-CB[8]-WGG, F'GG-CB[8]-FGG, F'GG-CB[8]-YGG and F'GG-CB[8]-NpGG complexes were identified with their calculated m / z values. Together, these data confirm the successful formation of new isopeptide dimers.

[0196] Isothermal titration calorimetry (ITC) was used to study the binding thermodynamics of isopeptide dimerization ( Figure 2 c, and additional data not shown). Titration of F'GG (3.0 mM) into CB[8] (0.1 mM) yielded a stepwise binding curve with two transitions at molar ratios of 1.0 and 2.0 ( Figure 2 c). The first step binding constant (K1) is 6.6x 10 5 M -1 , and the second step (K2) is 9.4x 10 3 M -1 This indicates that negative cooperativity exists when K1>K2 18,19,23 This favorable 1:1 complex enables secondary access to electron-rich aromatic peptides. Four binding curves were obtained by titrating XGG (X = W, F, Y, Np; 3.0 mM) into F'GG-CB[8] (0.2 mM), with a clear shift at the 1.0 molar ratio ( Figure 2 d), indicating that XGG was successfully incorporated into F'GG-CB[8].

[0197] Table 1 shows that all XGG peptides exhibited high binding strength (K a >10 4 M -1 ), confirming the thermodynamic stability of isopeptide dimerization. Figure 2 d, green) shows a relatively low Ka because the para-substituted hydroxyl group may reduce the enthalpy contribution and weaken the second association. 24 However, the overall binding constant (K1 Ka) of the isopeptide dimer F'GG-CB[8]-XGG was higher than 10 10 M -2 . With their parent dimers (e.g. F'GG-WGG, 25,26 K 二聚体 1M -1 ) showed a significant enhancement compared to the F'GG-CB[8]-FGG (heterodimer 2.4 x 10 11 M -2 ) of the total K1 K aHigher than 2FGG-CB[8] (homodimer, 1.5x 10 11 M -2 ) 6 Notably, no secondary association was observed for the nonaromatic analogs (KGG, EGG, LGG), highlighting the selectivity for aromatic peptides over nonaromatic peptides.

[0198] To understand the effect of the aromatic position on heterodimerization, the Ka of GXG and GGX (X = W, F, Y, Np) with F'GG-CB[8] was determined by ITC. Shifting the aromatic residue from the N-terminus to the C-terminus resulted in a significant decrease in Ka, see Table 1. The increased distance between the positive charge at the N-terminus and the aromatic motif weakened the ion-dipole interaction at the entrance of CB[8] and reduced the secondary binding affinity. Taking GGY as an example, no secondary binding with F'GG-CB[8] was observed. The seven new derivative isopeptide dimers, F'GG-CB[8]-GXG and F'GG-CB[8]-GGX, expanded the scope of host-enhanced isopeptide dimerization. The binding selectivity exhibited for aromatic residues is an advantage of this system, enabling the acquisition of a range of peptides and proteins. Compared with previous reports on CB[8]-peptide heteroternary complexes, 27 In contrast, the system described here is based only on F' amino acids and is easily linked in chemical biology and biochemistry.

[0199] Table 1: Thermodynamic data for the secondary association of 1:1 complexes of tripeptides containing aromatic residues and control tripeptides with F'GG-CB[8] a

[0200]

[0201]

[0202] Notes: a The average value was taken from three replicates. b Not detected.

[0203] Thermodynamic mechanisms behind peptide heterodimerization are attributed to social self-sorting, Figure 2 e, consistent with previous reports. 18,19Two pathways exist in an equimolar mixture of F'GG, CB[8], and XGG: social self-sorting (heterodimerization) and narcissistic self-sorting (homodimerization). Compared to homodimerization (Table S1), social self-sorting is favored in the presence of a certain ratio of isopeptide dimers and homopeptide dimers (e.g., WGG, R=2291), confirming quantitative isopeptide dimerization in the complex mixture. Notably, simply mixing the two aromatic tripeptides with CB[8] did not result in quantitative heterodimerization (data not shown).

[0204] After elucidating the thermodynamics, we demonstrate the utility of isopeptide dimers for on-resin recognition of aromatic peptides. Figure 1 c. Confocal fluorescence imaging was used to probe the interface recognition by a fluorescently labeled peptide (WGGGGG-dansyl), 9 Figure 3 a to b. A buffered solution of WGGGGG-dansyl-CB[8] (10 mM) was mixed with F'GGGGG-functionalized ChemMatrix resin (35-100 mesh particle size, surface loading = 0.5 mmol / g) under vigorous shaking at 25° C. for 10 min. Confocal fluorescence images were captured under gray field after laser irradiation (λ = 405 nm). Figure 3 cf showed that the resin with F'GGGGG-CB[8]-WGGGGG-dansyl showed significantly higher fluorescence than F'GGGGG, F'GGGGG-CB[8]-WGG, and F'GGGGG-WGGGGG-dansyl (data not shown), indicating successful isopeptide dimerization on the resin.

[0205] UV experiments were performed to test the absorption efficiency by quantifying the presence of aromatic peptides before and after treatment on the resin. Figure 4 a. A typical experiment involves mixing WGG-CB[8] (1.0 mM) with F'GGGGG-resin (10.0 mM) at 25 °C for 10 min. The absorbance intensity of the resin-treated WGG-CB[8] (grey) shows a decrease compared to the original (purple), Figure 4 b. The absorption efficiency of recognition on the resin was 77%, while recognition of free WGG by physical absorption was only 19% (data not shown). The absorbed WGG was released and recovered by competitive binding of memantine hydrochloride (DMADA) to regenerate the resin (additional data not shown).

[0206] Perform multiple cycles of resin identification to assess recyclability, Figure 4c (and additional data are not shown). The recognition and regeneration experiment of WGG-CB[8] was repeated for 3 cycles using the same batch of resin. Due to the complete release of WGG without any residue accumulation, the recovery efficiency on the resin remained above 98% over multiple cycles ( Figure 4 c). This confirmed the regeneration of the F'GGGGG-functionalized resin, making the entire process highly sustainable for practical use. We further investigated the selective separation of aromatic peptides by three cycles of recognition-release experiments using a peptide mixture of WGG, KGG, EGG, and LGG ([XGG] = 1.0 mM) in the presence of 1.0 mM CB[8] ( Figure 4 d, and Table 4 and the related discussion below, together with Figure 8 After 3 cycles, the ratio of WGG (purple) in the mixture increased from 26% to >95% ( Figure 4 d). Alternatively, residual DMADA and DMADACB[8] can be removed by liquid chromatography. This facile strategy to obtain aromatic peptides with high purity by on-resin isopeptide dimerization is easily automated.

[0207] To expand the applicability of this approach, we exploited interfacial recognition for insulin stabilization and its on-demand release from the resin. Figure 4 e and Table 2 (see below). Insulin is a widely used biological drug for the treatment of diabetes; 28 However, due to limited stability, it requires strict storage conditions (e.g., 2-6°C) as it readily forms immunogenic fibrillar aggregates in solution. 29 Insulin has an N-terminal phenylalanine that can serve as a guest.

[0208] UV experiments quantified the absorbance and on-demand release of insulin onto the resin. The insulin uptake efficiency was calculated to be 94%, and the intensity of its absorption decreased after treatment with the F'GGGGG-CB[8] resin. Figure 4 f and Figure 6 (See also Figure 5 UV spectrum of insulin on resin). Through competitive binding, insulin was displaced by DMADA with a recovery efficiency of 95% within 8 days of storage at room temperature ( Figure 4 g), indicating long-term stability ( Figure 7 and Table 3, shown below). Our method provides a way to store insulin under ambient conditions, eliminating the current need for refrigeration.

[0209] In summary, we introduce a new type of quantitative isopeptide dimerization. The binding affinity between the aromatic groups from two different peptides and the perfluorophenyl groups is significantly enhanced through host-enhanced polar interactions, and K a Up to 10 5 M-1 , and K1 K a Up to 10 11 M -2 , ensuring exclusive formation of isopeptide dimers. To demonstrate practicality, the solution-phase host-guest complex (F'GG-CB[8]-XGG) was transferred to a solid-liquid interface, enabling on-resin identification and separation of aromatic peptides as well as stabilization and on-demand release of insulin under ambient conditions. This general approach enables the accumulation and separation of aromatic-rich biomacromolecules useful in biomedical research. We anticipate that this work will inspire research on exploiting isopeptide dimerization as a versatile strategy for a wide range of life science applications.

[0210] Materials and instruments

[0211] Material

[0212] Unless otherwise noted, all starting materials were purchased from commercial suppliers and used without further purification: (L)-Fmoc-Glu(OtBu)-OH, (L)-Fmoc-Gly-OH, (L)-Fmoc-Leu-OH, (L)-Fmoc-Lys(Boc)-OH, (L)-Fmoc-Phe-OH, (L)-Fmoc-Trp(Boc)-OH, (L)-Fmoc-Tyr(tBu)-OH, (L)-Fmoc-3-(2-naphthyl)-alanine-OH, piperidine, diethyl ether, (L)-Fmoc-pentafluorophenylalanine-OH, diisopropylethylamine (DIPEA). =DIPEA), trifluoroacetic acid (TFA), triisopropylsilane (TIPS), N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), dimethylformamide (DMF), dichloromethane (DCM), sodium dihydrogen phosphate (NaH2PO4), sodium dihydrogen phosphate (Na2HPO4), heavy water (D2O, D, 99.8%), 1,3-dimethylaminoadamantane hydrochloride (memantine hydrochloride, DMADA), Rink amide-AM resin, H-Rink amide ChemMatrix resin (loading about 0.5 mmol / g functionalized surface), insulin (human recombinant). Phosphate buffer solution was prepared by adding 10 mM Na2HPO4 solution to 10 mM NaH2PO4 solution until pH was equal to 7.0. Cucurbitacin[8]uril (CB[8]) was synthesized and purified according to a previous report (see Kim), and the molecular weight of CB[8] was calibrated to 1600 g / mol. Water was obtained from a Milli-Q integrated water purification system (22.5 MΩ·cm).

[0213] Peptide synthesis and purification

[0214] All tripeptides (XGG, GXG, GGX) were synthesized on an automated microwave peptide synthesizer (Liberty Blue, CEM) using standard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Peptides were cleaved from Rink amide AM resin with a mixture of 95% TFA, 2.5% TIPS, and 2.5% H2O and shaken at room temperature for 1 hour. The resin was filtered and washed with the cleavage mixture. After evaporating most of the cleavage mixture, the peptides were crushed and washed three times with cold ether. Reverse phase high performance liquid chromatography (RP-HPLC) was used on a 5 μm pore size, The crude peptide was purified on a Phenomenex C18 Kinetic-Evo column with a particle size of 150×21.2 mm. The mobile phase of water contained 0.1% TFA. The purification method gradient was set from 5% acetonitrile and 95% water to 100% acetonitrile. The dry peptide (in the form of a TFA salt) was obtained by lyophilization and then verified by NMR and ESI-MS. Using the same method described above, the peptide F'GGGGG on resin was synthesized using H-Rink amide ChemMatrix resin. The F'GGGGG-functionalized resin was washed 3 times with DMF and then left in D2O / H2O (for 3 days, with fresh D2O / H2O replaced every day). The washed F'GGGGG resin was then stored in D2O / H2O in a refrigerator at 277K.

[0215] Nuclear Magnetic Resonance Spectroscopy (NMR)

[0216] The 1H, 2H and 3H spectra of the pure peptides were obtained in D2O at 298 K on a Bruker AVANCE 400 (400 MHz) instrument and a Bruker AVAENCE 500 instrument with a TCI cryoprobe (500 MHz). 13 C and 19 F NMR spectra. Other experiments (NMR titration, on-resin identification tests) were acquired in phosphate buffer (D2O) at pH 7.0 on a Bruker AVANCE400 (400 MHz) instrument. 1 H and 19 F NMR spectrum. Chemical shifts were referenced to the residual solvent peak of HDO at 4.79 ppm.

[0217] Isothermal Titration Calorimetry (ITC)

[0218] ITC experiments were performed at 298 K on a Malvern MicroCal Auto-ITC200 apparatus in 10 mM phosphate buffer, pH 7.0. A single injection of 0.6 μL and 32 consecutive injections of 1.2 μL were performed with 90 s intervals between each injection. The first data point was removed before analysis. The ITC data were fitted by the MicroCal Analysis Center software using a set of site models. In a typical experiment of homoternary complexation, the host molecule (CB[8]) was loaded in the sample cell at a concentration of 0.1 mM and the guest peptide was loaded in the syringe at a concentration of 2.0 mM. The injection and interval settings were the same as described above. The first data point was removed from the data set before analysis. The obtained ITC curves were fitted by the MicroCal Analysis Center software using a continuous binding model. All parameters were averaged with standard deviations using three replicates. n=3. Ka values ​​<10 2 M -1 Too low to be detected.

[0219] Electrospray ionization mass spectrometry (ESI-MS)

[0220] ESI-MS data were acquired on a Thermo Fisher Q Exactive Orbitrap mass spectrometer with a nanospray ion source. The positive mode was selected for all experiments at an operating temperature of 320 °C and a capillary voltage of 1.5 kV. All sample solutions were prepared in pure water.

[0221] Ultraviolet (UV) Spectroscopy

[0222] UV spectra were obtained on a Cary 4000 UV-Vis spectrometer. A pair of quartz cells with a 2 mm gap was used. Dual beam mode with a fixed 2 nm spectral bandwidth and baseline correction was selected. In a typical run, 300 μl of 1 mM solution was transferred to the quartz cell and scanned compared to phosphate buffer in D2O. For better quantification, UV spectra were obtained in the linear absorption range (0.2-0.8).

[0223] Confocal microscopy

[0224] Imaging on resin was performed on a Leica Stellaris 5 confocal microscope (Leica Microsystems), which had a 40x oil immersion objective and a numerical aperture of 1.3. The suspension of resin beads was dripped on a cover glass, and 50 images with a resolution of 1024x 1024 pixels were obtained at _EXC 405nm. The stacking boundary was selected at a point where the signal-to-noise ratio of the two extremes (edges) of the beads was not significantly different. The 3D data set was processed by Fiji / ImageJ, and maximum intensity Z projection was obtained to compare the mean fluorescence intensity of the resin beads. Three groups of resin beads were tested: Group A, original unmodified ChemMatrix resin (resin) and F'G5 functionalized resin (resin-G5F'); Group B, Resin-G5F' with WGG but without CB[8] (resin-G5F'+WGG), Resin-G5F' with WGG and CB[8] (resin-G5F'+WGG+CB[8]), and Resin-G5F' with WGG, CB[8] and then DMADA solution (resin-G5F' +WGG+CB[8]+DMAMA); Group C, Resin-G5F' with WG5-dansyl but without CB[8] (Resin-G5F'+WG5-dansyl), Resin-G5F' with WG5-dansyl and CB[8] (Resin-G5F'+WG5-dansyl+CB[8]), Resin-G5F' with WG5-dansyl, CB[8] and then DMADA solution (Resin-G5F'+WG5-dansyl+CB[8]+DMAMA). Three groups of resin beads were prepared according to the procedure in the UV spectrum above. All samples were then drained and the same amount of phosphate buffer at pH 7.0 was added to obtain a suspension (10 mM) for imaging.

[0225] Circular dichroism (CD) spectrum

[0226] CD measurements were performed on a Chirascan spectrometer (Applied Photophysics). Scans were performed at 298K from 200-500nm at 1.0nm intervals. In a 0.1cm path length quartz absorption cell, the acquisition time was 0.5 seconds. CD spectra were collected using solutions from the UV experiments of the above-mentioned 8-day storage and release experiments. The original insulin solution was 0.2mM. All solutions were diluted 2 times before measurement. All scans were collected and averaged (with background noise correction) in three scans.

[0227] Procedure for identification on resin

[0228] The standard procedure for NMR quantification of on-resin identification within a peptide mixture is as follows. A mixed peptide solution containing WGG, KGG, LGG, EGG, CB[8] ([XGG] = CB[8] = 1 mM) was used as the original cycle 0 solution. For cycle 1, 1.0 ml of mixed peptide (1 mM) was added to F'GGGGG-functionalized resin (10 mM) and shaken at room temperature for 10 min. After draining, 1 ml of 2 mM DMADA solution was added, shaken for 45 min, and collected as the released solution. For cycle 2, fresh CB[8] (2.88 mg, 1.8 μmol) was added to 0.9 ml of the solution released from cycle 1 (2 mM DMADA, 1.8 μmol) to combine with excess DMADA and separated WGG. About 0.1 ml of solution was lost after each cycle. The resin was added to this new solution, shaken for 45 min, drained, and then 0.9 ml of 2 mM DMADA solution was added to generate the solution released from cycle 2. For cycle 3, fresh CB[8] (2.56 mg, 1.6 μmol) was added to 0.8 ml of the solution released from cycle 2 (2 mM DMADA, 1.6 μmol). The resin was added to this new solution, shaken for 45 min, drained, and then 0.8 ml of the DMADA solution was added to produce the solution released from cycle 3. The original cycle 0 solution and the subsequent solutions released from each cycle were tested by NMR. After NMR analysis, the relative amount of WGG was determined by integrating a set of doublets at 3.36 ppm; the relative amount of KGG was determined by integrating a set of triplet peaks at 3.02 ppm; and the relative amount of EGG was determined by integrating a set of triplet peaks at 2.37 ppm. KGG, LGG, and EGG have an overlapping singlet peak at 3.95 ppm. Therefore, the relative amount of LGG was determined by subtracting the relative amounts of KGG and EGG from the peak integral at 3.95 ppm.

[0229] The standard procedure for UV quantification of recognition on peptide resin is as follows. Using the model tripeptide WGG as an example, a resin D2O suspension of 10 μmol F'GGGGG equivalents is added to a 12 ml syringe (r = 8.0 mm, h = 2.0 mm) with a polyester glass frit. After draining the D2O, 1 ml of 1 mM WGG:CB[8] = 1: 1 solution (original solution, in D2O, phosphate buffer, pH = 7.0) is added to the resin. Then, the piston and stopper are assembled to the syringe to form an airtight chamber. The syringe is transferred to a wrist oscillator and oscillated for 10 min. Then, the liquid in the syringe (resin-treated solution) is thoroughly drained, and the resin is washed once with D2O. Afterwards, 1 ml of 2 mM DMADA solution (in D2O, phosphate buffer, pH = 7.0) is added to the resin and oscillated for another 45 min. The solution (released solution) is drained for further testing. For 3 cycles of efficiency testing ( Figure 4 c), WGG:CB[8]=1:1 solution and DMADA solution were alternately added to the resin, shaken and drained to perform three cycles, and a series of resin-treated and released solutions were produced.

[0230] The standard procedure for UV quantification of insulin recognition on resin is as follows. Insulin (human recombinant) is suspended to 0.2 mM using 0.22 μm filtered H2O. 1N HCl is immediately added to reach an acid concentration of 5 mM. The solution is gently stirred until completely dissolved, filtered through a 0.22 μm filter, and stored at 277 K. The method is slightly modified from the cold spring harbor protocol (see recipe: insulin). The insulin solution is heated to room temperature before being used for recognition on the resin. In a typical experiment, 8 μmol F'GGGGG equivalents of the resin H2O suspension are added to a 12 ml syringe (r = 8.0 mm, h = 2.0 mm) with a polyester glass frit. After draining the H2O, 8 ml of 0.1 mM CB[8] solution is added to the resin, and the piston and stopper are assembled to the syringe to form an airtight chamber. The syringe is transferred to a wrist oscillator and shaken for 12 hours. Then, after draining CB[8] solution and washing the resin with H2O once, 0.8ml of 0.2mM insulin solution was added to the resin and vibrated gently for 3 hours. Then, the liquid in the syringe (resin-treated solution) was thoroughly drained and washed with H2O resin once. Afterwards, 0.8ml of 2mM DMADA solution (in 5mM HCl) was added to the resin and vibrated gently for 3 hours again. The solution (released solution) was drained. Immediately, the original insulin solution, the resin-treated solution and the released solution were measured on the UV spectrum.

[0231] The 8-day storage and release experiment was performed as follows. Each of the 9 syringes was prepared with a resin H2O suspension equivalent to 8 μmol F'GGGGG. On day 0, as a standard procedure for UV quantification of identification on the above insulin resin, all 9 syringes were drained, combined with CB[8], washed, combined with insulin, drained (to obtain a resin-treated solution), and washed. All syringes containing resin were kept at room temperature. From day 0 to day 8, 0.8 ml of a 2 mM DMADA solution was added to the syringe containing resin every day (to obtain a released solution, once a day). All resulting solutions were immediately analyzed on the UV spectrum. Figure 4 The numbers in g to calculate the percentage of release are the absorbance at 276 nm.

[0232] Synthesis and characterization of model peptides

[0233] (S)-2-amino-N-(2-((2-amino-2-oxoethyl)amino)-2-oxoethyl)-3-(perfluorophenyl)acrylamide (F'GG)

[0234] 1 H NMR (500MHz, D2O, ppm): δ = 4.32 (t, 1H), 3.99 (q, 2H), 3.93 (s, 2H), 3.37 (d, 2H); 13 C NMR (125MHz, D2O): δ=174.01,170.89,168.81,146.39,144.44,141.90,139.89,138.49,136.49,107.29,52.04,42.30,41.89,23.85; 19 F NMR (375 MHz, D2O, ppm): δ = -142.65, -154.41, -162.18; ESIMS for [MH]+: calcd. m / z = 369.10, found m / z = 369.17.

[0235] (S)-N-(2-amino-2-oxoethyl)-2-(2-aminoacetylamino)-3-(perfluorophenyl)propionamide (GF'G)

[0236] 1 H NMR (500MHz, D2O, ppm): δ = 4.77-4.74 (m, 1H), 3.95-3.86 (m, 2H), 3.85-3.79 (m, 2H), 3.32 (dd, 1H), 3.20 (dd, 1H); 13C NMR (125MHz, D2O, ppm): δ = 173.56, 172.10, 166.91, 146.25, 144.32, 141.27, 139.28, 138.29, 136.33, 109.48, 52.55, 42.00, 40.22, 24.43; 19 FNMR (375 MHz, D2O, ppm): δ = -143.48, -155.99, -162.89; ESIMS of [MH]+: calculated value m / z = 369.10, measured value m / z = 369.17.

[0237] (S)-2-(2-(2-aminoacetamido)acetamido)-3-(perfluorophenyl)propionamide (GGF')

[0238] 1 H NMR (500MHz, D2O, ppm): δ = 4.73-4.70 (m, 1H), 3.96 (s, 2H), 3.88 (s, 2H), 3.31 (dd, 1H), 3.16 (dd, 1H); 13 C NMR (125MHz, D2O, ppm): δ = 174.32, 170.86, 167.64, 146.27, 144.34, 141.17, 139.17, 138.24, 136.27, 109.89, 52.04, 42.05, 40.28, 24.52; 19 F NMR (375 MHz, D2O, ppm): δ = -143.47, -156.31, -163.04; ESIMS for [MH]+: calcd. m / z = 369.10, found m / z = 369.17.

[0239] (S)-2-Amino-N-(2-((2-amino-2-oxoethyl)amino)-2-oxoethyl)-3-(1H-indol-3-yl)propanamide (WGG)

[0240] 1 H NMR (500MHz, D2O, ppm): δ = 7.64 (d, 1H), 7.54 (d, 1H), 7.35 (s, 1H), 7.29 (t, 1H) ,7.21(t,1H),4.36(t,1H),3.92(d,1H),3.91-3.75(m,3H),3.49-3.38(m,2H); 13C NMR (125 MHz, D2O, ppm): δ = 173.90, 171.22, 170.24, 136.11, 126.42, 125.23, 122.21, 119.61, 117.98, 112.03, 106.24, 53.68, 42.36, 41.83, 26.70; ESIMS for [MH]+: calcd. m / z = 318.16, found. m / z = 318.34.

[0241] (S)-N-(2-amino-2-oxoethyl)-2-(2-aminoacetylamino)-3-(1H-indol-3-yl)propionamide (GWG)

[0242] 1 H NMR (500MHz, D2O, ppm): δ = 7.67 (d, 1H), 7.52 (d, 1H), 7.30-7.24 (m, 2H), 7.19 (t, 1H) ,4.77-4.69(m,1H),3.84(d,1H),3.81-3.73(m,2H),3.70(d,1H),3.36-3.23(m,2H); 13 C NMR (125 MHz, D2O, ppm): δ = 173.90, 173.77, 166.95, 136.04, 126.64, 124.54, 122.00, 119.38, 118.25, 111.89, 108.60, 54.89, 42.00, 40.23, 26.96; ESIMS for [MH]+: calcd. m / z = 318.16, found. m / z = 318.34.

[0243] (S)-2-(2-(2-aminoacetamido)acetamido)-3-(1H-indol-2-yl)propanamide (GGW)

[0244] 1 H NMR (500MHz, D2O, ppm): δ = 7.70 (d, 1H), 7.52 (d, 1H), 7.30-7.24 (m, 2H), 7.19 (t, 1H),4.68(dd,1H),3.99-3.87(m,2H),3.77(s,2H),3.34(dd,1H),3.23(dd,1H); 13 C NMR (125MHz, D2O, ppm): δ = 176.11, 170.80, 167.56, 136.05, 126.84, 124.49,

[0245] 121.89, 119.30, 118.29, 111.84, 108.86, 54.13, 42.11, 40.23, 26.96; ESIMS of [MH]+: calculated value m / z=318.16, measured value m / z=318.34.

[0246] (S)-2-amino-N-(2-((2-amino-2-oxoethyl)amino)-2-oxoethyl)-3-phenylpropanamide (FGG)

[0247] 1 H NMR (500MHz, D2O, ppm): δ = 7.47-7.36 (m, 3H), 7.35-7.29 (m, 2H), 4.32 (t 1H), 4.01 (d, 1H), 3.94-3.87 (m, 3H), 3.29-3.20 (m, 2H); 13 C NMR (125 MHz, D2O, ppm): m / z = 173.97, 171.27, 169.82, 133.68, 129.35, 129.14, 128.00, 54.41, 42.26, 41.90, 36.69; ESIMS for [MH]+: calculated value m / z = 279.15, measured value m / z = 279.34.

[0248] (S)-N-(2-amino-2-oxoethyl)-2-(2-aminoacetylamino)-3-phenylpropionamide (GFG)

[0249] 1 H NMR (500MHz, D2O, ppm): δ=7.44-7.37(m,2H),7.37-7.31(m,1H),7.34-7.28(m, 2H),4.68(t,1H),3.86(t,2H),3.81-3.74(m,2H),3.16(dd,1H),3.07(dd,1H); 13 C NMR (125 MHz, D2O, ppm): δ = 173.76, 173.49, 166.96, 136.05, 129.14, 128.80, 127.28, 55.32, 41.99, 40.18, 36.93; ESIMS for [MH]+: calcd. m / z = 279.15, found. m / z = 279.34.

[0250] (S)-2-(2-(2-aminoacetamido)acetamido)-3-phenylpropanamide (GGF)

[0251] 1H NMR (500MHz, D2O, ppm): δ = 7.43-7.36 (m, 2H), 7.36-7.29 (m, 2H), 7.30 (t, 1H), 4.62 (dd, 1H), 3.94 (q, 2H), 3.85 (s, 2H), 3.19 (dd, 1H), 3.01 (dd, 1H); 13 C NMR (125 MHz, D2O, ppm): δ = 175.70, 170.82, 167.57, 136.41, 129.14, 128.69, 127.11, 54.68, 42.01, 40.29, 36.98; ESIMS for [MH]+: calculated value m / z = 279.15, measured value m / z = 279.34

[0252] (S)-2-(2-(2-aminoacetylamino)acetylamino)-3-(4-hydroxyphenyl)propionamide (GGY)

[0253] 1 H NMR (500MHz, D2O, ppm): δ = 7.20-7.13 (m, 2H), 6.90-6.83 (m, 2H), 4.56 (dd, 1H), 3.95 (q, 2H), 3.86 (s, 2H), 3.10 (dd, 1H), 2.94 (dd, 1H); 13 C NMR (125 MHz, D2O, ppm): δ = 175.77, 170.79, 167.57, 154.37, 130.49, 128.22, 115.38, 54.84, 42.01, 40.30, 36.18; ESIMS for [MH]+: calcd. m / z = 295.14, found. m / z = 295.34.

[0254] (S)-2-amino-N-(2-((2-amino-2-oxoethyl)amino)-2-oxoethyl)-3-(naphthalen-2-yl)propionamide (NpGG)

[0255] 1 H NMR (500MHz, D2O, ppm): δ = 7.97 (t, 2H), 7.95-7.90 (m, 1H), 7.83-7.79 (m, 1H), 7.64-7.55 (m, 2H) ,7.46(dd,1H),4.44-4.39(m,1H),3.89(s,2H),3.74-3.62(m,2H),3.44(dd,1H),3.37(dd,1H); 13C NMR (125 MHz, D2O, ppm): δ = 173.74, 171.04, 169.72, 133.04, 132.38, 131.40, 128.84, 128.33, 127.72, 127.62, 126.92, 126.84, 126.61, 54.27, 42.23, 41.68, 36.87; ESIMS for [MH]+: calcd. m / z = 329.16, found. m / z = 329.34.

[0256] (S)-N-(2-amino-2-oxoethyl)-2-(2-aminoacetylamino)-3-(naphthalen-2-yl)propionamide (GNpG)

[0257] 1 H NMR (500MHz, D2O, ppm): δ = 7.93 (td, 3H), 7.80-7.76 (m, 1H), 7.57 (tt, 2H), 7.46 (dd ,1H),4.77-4.74(m,1H),3.84(dd,2H),3.73(dd,2H),3.32(dd,1H),3.23(dd,1H); 13 C NMR (125 MHz, D2O, ppm): δ = 173.67, 173.45, 166.94, 133.76, 133.04, 132.10, 128.31, 127.75, 127.62, 127.47, 127.24, 126.62, 126.21, 55.25, 41.96, 40.16, 37.13; ESIMS of [MH]+: calculated value m / z = 329.16, measured value m / z = 329.34.

[0258] (S)-2-(2-(2-aminoacetamido)acetamido)-3-(naphthalen-2-yl)propanamide (GGNp)

[0259] 1 H NMR (500MHz, D2O, ppm): δ = 7.97-7.89 (m, 3H), 7.79-7.75 (m, 1H), 7.57 (tt, 2H), 7. 46(dd,1H),4.73(dd,1H),3.90(q,2H),3.76(s,2H),3.36(dd,1H),3.16(dd,1H); 13C NMR (125 MHz, D2O, ppm): δ = 175.66, 170.78, 167.47, 134.17, 133.03, 132.06, 128.19, 127.71, 127.61, 127.49, 127.31, 126.51, 126.11, 54.53, 42.02, 40.21, 37.16; ESIMS for [MH]+: calcd. m / z = 329.16, found. m / z = 329.34.

[0260] (S)-2,6-Diamino-N-(2-((2-amino-2-oxoethyl)amino)-2-oxoethyl)hexanamide (KGG)

[0261] 1 H NMR (500MHz, D2O, ppm): δ = 4.12-4.02 (m, 3H), 3.95 (s, 2H), 3.02 (t, 2H), 2.01-1.90 (m, 2H), 1.73 (p, 2H), 1.55-1.43 (m, 2H); 13 C NMR (125 MHz, D2O, ppm): δ = 0.5, 25.74; ESIMS for [MH]+: calcd. m / z = 261.12, found m / z = 261.34.

[0262] (S)-2-amino-N-(14-amino-2,5,8,11,14-pentyloxy-3,6,9,12-tetraazatetradecyl)-3-(perfluorophenyl)acrylamide (F'GGGGG)

[0263] 1 H NMR (700MHz, D2O, ppm): δ = 4.30 (t, 1H), 4.02 (m, 8H), 3.95 (s, 2H), 3.37 (d, 2H); 19 F NMR (375 MHz, D2O, ppm): δ = -142.63, -154.54, -162.22; ESIMS for [MH]+: calcd. m / z = 540.16, found m / z = 540.67.

[0264] 2-Amino-N-(14-amino-2,5,8,11,14-pentyloxy-3,6,9,12-tetraazatetradecyl)acetamide (GGGGGG)

[0265] 1H NMR (700 MHz, D2O, ppm): δ = 4.09 (s, 2H), 4.04 (d, 4H), 4.02 (s, 2H), 3.95 (s, 2H), 3.93 (s, 2H); ESIMS for [MH]+: calculated value m / z = 360.16, measured value m / z = 360.50.

[0266] (S)-2-amino-N-(17-((5-(dimethylamino)naphthalene)-1-sulfonylamino)-2,5,8,11,14-pentoxy-3,6,9,12,15-pentaazaheptadecanyl)-3-(1H-indol-3-yl)acrylamide (WGGGGG-dansyl)

[0267] 1 H NMR (700MHz, D2O, ppm): δ = 8.68 (d, 1H), 8.47 (d, 1H), 8.36 (d, 1H), 7.97-7.85 (m, 3 H),7.62(d,1H),7.53(d,1H),7.35(s,1H),7.26(t,1H),7.17(t,1H),4.41(t,1H), 4.07 (s, 2H), 4.03 (s, 2H), 4.02-3.92 (m, 4H), 3.69 (s, 2H), 3.48-3.40 (m, 2H), 3.37 (s, 6H), 3.21 (t, 2H), 3.14 (t, 2H); ESIMS of [MH]+: calculated value m / z = 765.31, measured value m / z = 765.34.

[0268] On-resin identification of insulin

[0269] like Figure 5 As shown in a, without using CB[8] Figure 4 f Under the same experimental conditions, insulin showed minimal absorption on F'GGGGG-resin. Figure 5 In b, c, by using the same ChemMatrix resin but with a different peptide GGGGGG instead of F'GGGGG, insulin showed negligible absorption in both cases, regardless of the use of CB[8]. Figure 5 In Figure d, even when insulin and CB[8] were used simultaneously, the blank resin (i.e., purchased H-Rink amide ChemMatrix resin without any loaded peptide) showed almost no absorption. This confirms that the absorption of insulin-CB[8] on F'GGGGG-resin is achieved through the host-enhanced polar-π interaction.

[0270] A higher efficiency of 94% was observed when the F'GGGGG resin was treated with an insulin (0.2 mM) solution, compared to 77% efficiency when the F'GGGGG resin was treated with a WGG (1.0 mM) solution. This phenomenon is actually one of the advantages of insulin stabilization on the resin. Considering that the Ka values ​​of WGG and FGG are very close, the difference in absorption efficiency between WGG and insulin is not only related to equilibrium calculations. Unlike WGG, insulin has two relatively long peptide chains consisting of 51 amino acids, so its relatively large size can make it kinetically trapped in the cross-linked PEG (material of ChemMatrix resin) resin network. This can increase the absorption efficiency to about 94%, much higher than the free small tripeptide WGG.

[0271] Table 2: UV absorbance data at 276 nm for insulin stored on resin and released on demand on days 0, 2, 5, and 8 of an 8-day experiment

[0272]

[0273] Notes: a . Calculated from Table S8 using the following equation Figure 4 Efficiency results for g:

[0274]

[0275] Secondary structure verification of released insulin

[0276] like Figure 7 As shown in Table 3, after 2, 5, and 8 days of storage on the functionalized resin, all three samples showed highly similar CD curves when released back into the solution phase compared to the freshly prepared insulin solution. This indicates that the secondary structure of insulin is intact after 2, 5, and 8 days of storage at room temperature.

[0277] Table 3: CD absorbance data at 208 nm for insulin stored on resin and released on demand on days 2, 5, and 8 of an 8-day experiment

[0278]

[0279] Additional Experiments

[0280] Selective separation of aromatic peptides on resin

[0281] The released DMADA-CB[8] complex may have some weak interactions (i.e., electrostatic interactions, hydrogen bonds) with the remaining tripeptide, which may lead to some changes in chemical shifts after release. The yield of WGG from the selective separation of a single cycle was 77% (data not shown), so after 3 cycles of selective separation, the final yield of WGG was 77% × 77% × 77% = 45.6% (see also Figure 8 ).

[0282] Table 4: Peptide mixtures identified on the resin 1 H NMR comprehensive analysis a

[0283]

[0284]

[0285] Notes: a All the above percentage results are shown in Figure 4 d.

[0286] References

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Claims

1. A ternary complex of a cucurbituril host with a first aryl guest and a second aryl guest, wherein the first aryl guest is a group within a biomolecule, and the first aryl guest and the second aryl guest are different.

2. The complex according to claim 1, wherein the cucurbituril host is CB[8] or a variant or derivative thereof.

3. The complex of claim 2, wherein the cucurbituril host is CB[8].

4. The complex according to any one of the preceding claims, wherein the first aryl guest is selected from phenyl, naphthyl such as naphthalen-2-yl, hydroxyphenyl such as 4-hydroxyphenyl, and indolyl such as indol-3-yl.

5. The complex according to any one of the preceding claims, wherein the second aryl guest is a fluorophenyl group, such as a perfluorophenyl group.

6. The complex according to any one of the preceding claims, wherein the second aryl guest is covalently attached directly or indirectly to the solid phase.

7. The complex according to any one of the preceding claims, wherein the biomolecule is a polypeptide.

8. The complex according to any one of the preceding claims, wherein the first aromatic guest is provided within the side chain of a phenylalanine, tyrosine, indole or naphthylalanine residue.

9. The complex according to any one of the preceding claims, wherein the biomolecule is insulin, such as human insulin, such as recombinant human insulin.

10. The complex of claim 9, wherein the first guest is a phenyl group within the phenylalanine residue at position 1 of insulin.

11. A method of decomplexing a complex according to any one of claims 1 to 10, the method comprising the steps of treating the complex with a competing guest and allowing the competing guest to displace at least the first aromatic guest from the complex.

12. The method of claim 11, wherein the competing object is or comprises amantadine (ADA) or memantine (DMADA).

13. A method of forming a complex according to any one of claims 1 to 10, the method comprising the steps of mixing a cucurbituril host with a biomolecule having a first aryl guest and a second aryl guest, and allowing the host and guest to self-assemble.

14. A method according to claim 13, comprising the step of at least partially purifying the complex, such as by filtration.

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