Supramolecular complexes of biomolecules

Abstract

The present invention provides a ternary complex of a cucurbituril host, e.g., CB[8], with a first aryl guest and a second aryl guest, where the first aryl guest is a group in a biomolecule, such as insulin, and the first aryl guest and the second aryl guest are different. Also provided is a method of decomplexing the complex, comprising treating the complex with a competing guest and allowing the competing guest to displace at least the first aryl guest from the complex.

Description

[Technical field]

[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). Related Applications This application claims priority to and the benefit of GB2205916.6, filed on April 22, 2022 (22.04.2022), and GB2206222.8, filed on April 28, 2022 (28.04.2022), the contents of each of which are incorporated by reference in their entirety herein.

[0002] The present invention provides methods 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 technology]

[0003] Methods for improving the stability, and thus the shelf life, of biomolecules such as insulin are of interest in the medical field. Webber et al. explored the modification of insulin with poly(ethylene glycol) (PEG) to enhance the stability of the polypeptide. Here, insulin is incorporated into a cucurbit[7]uril (CB[7])-PEG conjugate. CB[7] is covalently attached to the PEG group. CB[7] forms a binary complex with the phenyl group of the Phe residue at position 1 of insulin. This host-guest complex is used as an essentially permanent bond to insulin in vivo, with the conjugate used directly in this form, and the authors propose that the presence of the CB complex confers advantages to its use in vivo, such as the potential advantages associated with delayed and controlled uptake for higher molecular weight insulins.

[0004] Webber et al. show that cucurbit[7]uril can be used to form complexes with other biomolecules, including glucagon and anti-CD20 antibodies. Summary of the Invention [Problem to be solved by the invention]

[0005] However, modification of biomolecules may not always be beneficial, and modifications may not always maintain or improve biological activity. There is a need for stabilization strategies that allow the recovery and use of biomolecules in their native form. [Means for solving the problem]

[0006] In a general aspect, the invention provides a ternary complex comprising a cucurbituril host bearing a first aryl guest and a second aryl guest, where the first guest is a biomolecular group such as a polypeptide.

[0007] The second guest is different from the first guest. Thus, the complex is a heterocomplex. The formation of a heterocomplex may be favored over the formation of a homocomplex, where the guest is the same. This type of complex may be beneficially used to stabilize biomolecules in storage, which can be easily and quickly released from the complex when required, thus limiting the degradation of the biomolecule and maintaining its activity upon release.

[0008] The selective capture of a biomolecule in a complex can also allow the biomolecule to be separated from other components in a mixture, thereby at least partially purifying the biomolecule, for example, the complex can be bound to a solid phase, allowing recovery of the complex by simple filtration and separation from other solubilized components of the mixture.

[0009] 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. The first aryl guest may be a phenyl group, such as that present in a phenylalanine residue of a polypeptide, which may be a terminal or mid-chain residue within the polypeptide, preferably a terminal residue, e.g., an amino acid residue in the N-terminal region of the polypeptide, such as the N-terminal residue.

[0010] The polypeptide may be a protein, including an antibody. The protein is preferably insulin, such as human insulin, including human recombinant insulin. The first guest may be, for example, the phenyl group of the phenylalanine residue (1-Phe) at position 1 of insulin in the B chain.

[0011] The second guest may be a perfluorophenyl group (i.e., pentafluorophenyl), which may be directly or indirectly covalently attached to a solid phase, such as a polymeric solid phase.

[0012] In a further aspect of the invention there is provided a method of forming a complex such as the complex of the first aspect of the invention comprising mixing a cucurbituril together with a biomolecule having a first aryl guest and a second aryl guest and allowing the host and guest to self-assemble.

[0013] The complex may be formed by self-assembly. The process may be, and preferably is, carried out under aqueous conditions. The process may be, and preferably is, carried out at ambient temperature, for example at a temperature in the range of 5-30°C.

[0014] In a further aspect, a method of decomplexing a complex of the invention is provided, comprising 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.

[0015] The process may be, and preferably is, carried out under aqueous conditions. The process may be, and preferably is, carried out at ambient temperature, for example at a temperature in the range of from 5 to 30°C. The decomplexing method results in the release of the biomolecule from the complex.

[0016] The competing guest may be a compound that is a competing guest or that contains a competing guest. In a preferred embodiment, the competing compound is memantine (DMADA), which may be used as memantine hydrochloride.

[0017] The release of the biomolecule from the complex may include a preliminary step of forming the complex, such as by the method of the present invention. The release of the complex may be carried out one week or more after formation, for example one month after formation. The complex may be stable and prevent the degradation of the biomolecule. Therefore, the complex may be suitable for long-term storage of the biomolecule, which may be released from the complex when necessary without significant loss of biological activity.

[0018] These and other aspects and embodiments of the invention are described in detail below. [Brief description of the drawings]

[0019] [Figure 1] a) Host-enhanced heteropeptide dimers formed from F'GG, WGG, and CB[8]; b) molecular structures of FGG, YGG, and NpGG; c) schematic of on-resin recognition via interfacial heterodimerization. [Figure 2-1] a) H and F NMR spectra (DO, 298 K) obtained via titration of WGG (16.0 mM) into F'GG-CB[8] (1.0 mM); b) HR ESI-MS spectra (HO, 1.0 mM) of the 1:1 complex of F'GG-CB[8] and the heteropeptide dimer consisting of WGG, FGG, YGG, and NpGG; c) ITC titration plot of F'GG (3.0 mM) into CB[8] (0.1 mM) (10 mM phosphate buffer, pH 7.0, 298 K); d) ITC titration plot of WGG, FGG, YGG, and NpGG (3.0 mM) into F'GG-CB[8] (0.2 mM) (10 mM phosphate buffer, pH 7.0, 298 K); and e) schematic of the self-sorting mechanism. [Figure 2-2] Figure 2 continued. [Diagram 3] a) Molecular structure of WGGGGG-dansyl and b) Schematic of interface recognition with F'GGGGG resin (10 mM), c) F'GGGGG resin obtained via 405 nm laser excitation under the grey area, and confocal fluorescence images of it plus d) WGG-CB[8] (10 mM), e) WGGGGG-dansyl (10 mM), and f) WGGGGG-dansyl-CB[8] (10 mM). Scale bar = 200 m. Fluorescence intensity is quantified by mean grey value (MGV). [Figure 4-1]a) Schematic of on-resin recognition 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) Histogram of the refolding efficiency of WGG in successive on-resin recognition cycles; d) Histogram of the percentage of WGG obtained after multiple cycles of isolation; e) Schematic, not drawn to scale, of on-resin stabilization, on-demand release, and binding of insulin to CB[8]; f) UV spectra of insulin (0.2 mM) before and after treatment with F'GGGGG-CB[8]-resin (10.0 mM) followed by release at 0, 2, 5, and 8 days; g) Histogram of the refolding efficiency of insulin at 0, 2, 5, and 8 days. [Figure 4-2] Figure 4 continued. [Diagram 5] Figure 1 shows UV spectra of control experiments for on-resin recognition of insulin (0.2 mM): a) on-resin recognition of insulin (0.2 mM) on F'GGGGG-resin (10 mM), b) on-resin recognition of insulin (0.2 mM) with CB[8] (1.0 mM) on GGGGGG-resin (10 mM), c) on-resin recognition of insulin (0.2 mM) on GGGGGG-resin (10 mM), d) on-resin recognition of insulin (0.2 mM) with CB[8] (1.0 mM) on blank resin (H-Rink Amide ChemMatrix resin) (phosphate buffer, HO). [Figure 6] FIG. 1 shows UV spectra of insulin (0.2 mM) solution before and after treatment with F'GGGGG-CB[8]-resin (10.0 mM) followed by time-dependent release (phosphate buffer, HO) at 0, 2, 5, and 8 days. [Figure 7] FIG. 13 shows CD spectra of insulin solutions 2, 5, and 8 days after treatment with F'GGGGG-CB[8]-resin and subsequent time-dependent release (phosphate buffer, HO). [Figure 8]FIG. 1H NMR of selective on-resin isolation of aromatic peptides from a mixed peptide mixture (WGG, KGG, EGG, LGG 1.0 mM, phosphate buffer, DO) over three cycles using F'GGGGG-resin (10 mM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present inventors have found that a CB[8] host can be used to form a complex with a biomolecule, such as a polypeptide, together with a second component selected for heteropairing with a group of the biomolecule in the cavity of the host.

[0021] This complex has been found to stabilize biomolecules such as insulin for storage, which can be released on demand for use, without compromising the integrity of the biomolecule, including its structural and functional integrity.

[0022] Li et al. describe the use of CB[7] to form binary complexes with insulin or human growth hormone (hGH), where CB[7] is covalently attached to a solid phase. Insulin and HGA can be released from the complexes by treatment with competing guests. However, Li et al. do not show or suggest that the formation of the complexes is associated with stabilization of the insulin or hGH biomolecules.

[0023] Additionally, the preparation of CB[7] and its covalent attachment to solid phases is not straightforward and cumbersome, making accessibility and scalability problematic. In contrast, the use of the rapidly available and easily prepared CB[8] and related larger cucurbituril hosts as described herein avoids the problems associated with the use of CB[7] and its derivatives.

[0024] Complex The complex of the present invention comprises a cucurbituril host, e.g., CB[8], that non-covalently holds within its cavity a first guest and a second guest, which are supramolecular complexes and heterocomplexes, and the first and second guests are distinct.

[0025] Cucurbituril forms non-covalent bonds with each guest, and the inventors have previously established that cucurbituril complexes can be rapidly formed and used to create strong non-covalent bonds between compounds.

[0026] Complexes can be formed between the cucurbituril and a first guest from a biomolecule, and this complex formation is tolerant to multiple functionalities within the monomer. The complex also includes a second guest, and in a preferred embodiment of the invention, this second guest is bound to a solid phase, the formation of the complex being tolerant to the functionality of such solid phase.

[0027] When a complex contains two guests within the cucurbituril cavity, the binding 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 binding constant is at least 10 10 M -2 It is.

[0028] The cucurbituril hosts two distinct guest molecules, and the formation of the ternary host-guest complex is thought to proceed via an intermediate binary complex.

[0029] The reference herein to a first and second guest does not describe the order in which these guests complex with the cucurbituril host, and thus intermediate product binaries may be formed with the second guest and the host, or the first guest and the host.

[0030] Complex formation is reversible. Separation of the guest from the cucurbituril host is sometimes referred to as decomplexation. Decomplexation of the complex to separate the host and guest can occur, for example, as a response to external stimuli including competing guest compounds, light, oxidizing or reducing agents, electrochemical potential, and temperature changes, among others. In the methods of the present invention, decomplexation of the complex is carried out under conditions that will not damage or interfere with the structure of the biomolecules. For this reason, displacement methods involving harmless competing guest compounds are preferred.

[0031] Competitive guests for use in decomplexing CB[8]-based networks are or include adamantanamine (ADA) or memantine (DMADA). Competitive guests may be used in molar excess relative to the amount of guest present in the complex. In one embodiment, the competitive guest has a higher binding constant than the guest of the complex.

[0032] The decomplexation reaction is reversible, such that the complex may be allowed to reform, if desired, following removal of the external stimulus, including, for example, removal of a competing guest compound.

[0033] Cucurbituril and CB[8] The complexes of the present invention have a cucurbituril host that has the ability to host two guests, and thus the host may be a CB[8] host, including variants and derivative forms.

[0034] Cucurbiturils that have the ability to form ternary and binary complexes find use in the present invention. There are many examples in the field of cucurbiturils for use in forming such complexes.

[0035] Recent studies have shown that cucurbituril compounds have high in vitro and in vivo biocompatibility and extremely low toxicity (see Uzunova et al., Org. Biomol. Chem. 2010, 8, pp. 2037-2042). Therefore, when used with non-toxic polymeric components, the current hydrogels are also suitable for use in biological systems.

[0036] The cucurbituril should have the ability to form ternary complexes. For example, CB[8] has the ability to form ternary complexes, as do CB

[10] and CB

[12] compounds.

[0037] The cucurbituril may be a CB[8], CB

[10] , or CB

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

[0038] Reference to a cucurbituril compound is a reference to variants and derivatives thereof. Variants of CB[8] may include structures with one or more repeating units structurally similar to glycoluril. The repeating units may include ethylurea units. If all of the units are ethylurea units, the variant is a hemicucurbituril. A variant may be a hemicucurbit

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

[0039] [ka]

[0040] Cucurbituril derivatives are provided and used in the methods described herein. Derivatives of cucurbituril are structures having one, two, three, four or more substituted glycoluril units. Substituted cucurbituril compounds have the following structure:

[0041] [ka]

[0042] (In the formula, n is an integer of at least 8, For each glycoluril unit, Each X is O, S, or NR 3 and -R 1 and -R 2 is -H and the following optionally substituted groups: -R 3 , -OH, -OR 3 , -COOH, -COOR 3 , -NH2, -NHR 3 and -N(R 3 )2(in the formula, -R 3 is C 1~20 Alkyl, C 6~20 Carboaryl, and C 5~20 heteroaryl), or -R1 and / or -R 2 is -N(R 3 )2, and both -R 3 Let's get together and 5~7 Form a heterocyclic ring, or -R 1 and -R 2 together with the uracil skeleton to form C 6~8 C forms a carbocyclic ring 4~6 alkylene) It can be expressed as:

[0043] In one embodiment, one of the glycoluril units is a substituted glycoluril unit, so that for n-1 of the glycoluril units, -R 1 and -R 2 are each independently -H.

[0044] In one embodiment, n is 8, 9, 10, 11, or 12. In one embodiment, n is 8, 10, or 12. In one embodiment, n is 8.

[0045] In one embodiment, each X is O. In one embodiment, each X is S. In one embodiment, R 1 and R 2 are each independently H.

[0046] In one embodiment, for each unit, R 1 and R 2 one of which is H and the other is -H and the following optionally substituted group: -R 3 , -OH, -OR 3 , -COOH, -COOR 3 , -NH2, -NHR 3 , and -N(R 3 In one embodiment, for each glycoluril unit, R 1 and R2 one of which is H and the other is -H and the following optionally substituted group: -R 3 , -OH, -OR 3 , -COOH, -COOR 3 , -NH2, -NHR 3 , and -N(R 3 In this embodiment, the remaining glycoluril units are independently selected from R 1 and R 2 are each independently H.

[0047] Preferably, -R 3 is C 1~20 alkyl, most preferably C 1~6 It is an alkyl group. 1~20 The alkyl group may be linear and / or saturated. 3 The groups may be independently unsubstituted or substituted. Preferred substituents are -R 4 , -OH, -OR 4 , -SH, -SR 4 , -COOH, -COOR 4 , -NH2, -NHR 4 , and -N(R 4 )2(in the formula, -R 4 is C 1~20 Alkyl, C 6~20 Carboaryl, and C 5~20 The substituents are selected from -COOH and -COOR. 4 may be independently selected from

[0048] In some embodiments, -R 4 -R 3 In some embodiments, -R 4 is preferably unsubstituted. -R 1 and / or -R 2 But -OR 3 , -NHR 3 , or -N(R 3)2, then -R 3 is preferably C 1~6 In some embodiments, -R 3 is the substituent -OR 4 , -NHR 4 , or -N(R 4 )2. Each -R 4 is C 1~6 It is an alkyl, which is preferably itself substituted.

[0049] Cucurbituril Guest As mentioned above, the guest is a compound that has the ability to form a host-guest complex with cucurbituril.Therefore, the term complexation refers to the establishment of a host-guest complex.This host-guest complex is a ternary complex that includes the cucurbituril host, 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.

[0050] Typically, such complexes are based on CB[8] and its variants and derivatives. The first guest and the second guest are not the same. In principle, any compound with suitable binding affinity (as noted in the discussion of complexes above) may be used in the present invention. The compounds used may be selected based on the size of the moieties that are likely to interact with the cucurbituril cavity. The size of these moieties may be large enough to only allow complexation with larger cucurbituril forms.

[0051] In the present case, each of the first guest and the second guest is an aryl group. The aryl group may be a carboaryl group or a heteroaryl group, preferably a carboaryl group.

[0052] The aryl group may be a monocyclic, bicyclic, or tricyclic aryl group. The aryl group may be a monocyclic or a bicyclic aryl group. An aryl group can be unsubstituted or substituted, such as monosubstituted.

[0053] Examples of carboaryl groups include phenyl, naphthyl, and anthracenyl. Examples of heteroaryl groups include indolyl.

[0054] The first and second guests are not the same.In some embodiments, the aryl rings can be the same, but these guests differ in the number and / or identity of the substituents on the rings.For example, in this embodiment, the first guest of phenyl group is used together with the second guest having a phenyl ring, but this ring is perfluorinated.

[0055] The substituents of each aryl group are alkyl, halo, -OH, -OR 1 , -NH2, -NHR 1 , -NR 1 R 2 and -R 1 and -R 2 are each independently alkyl.

[0056] Alternatively, the substituents on each aryl group can be alkyl, halo, -OH, -OR 1 , -CN, -NO2, -NH2, -NHR 1 , -NR 1 R 2 and -R 1 and -R 2 are each independently alkyl.

[0057] The alkyl group is C 1~6 Alkyl, e.g. C 1~4 It may be alkyl, such as methyl or ethyl, such as methyl. The halo group may be selected from -F, -Cl, -Br, and -I, and is preferably -F.

[0058] Typically, one aryl guest is halogenated, such as fluorinated, while the other aryl guest is not halogenated. It is preferred that the second aryl guest is halogenated, such as fluorinated. Here, it is also preferred that the first aryl guest is not halogenated, such as not fluorinated.

[0059] The present inventors have found that electron-deficient guests, such as halogenated aryl guests, such as perfluorphenyl, advantageously form heterocomplexes with electron-rich guests, such as substituted aryl guests.

[0060] Typically, the first guest is an unsubstituted or monosubstituted aryl group. Typically, the second guest is a substituted aryl group.

[0061] The first guest may be selected from the group consisting of phenyl, naphthyl, such as naphth-2-yl, hydroxyphenyl, such as 4-hydroxyphenyl, and indolyl, such as indol-3-yl.

[0062] The first aryl guest may be covalently bonded to the methylene group. The first guest may be an indolyl, including a phenyl or an indol-3-yl.

[0063] Preferably, the first guest is a phenyl. In another embodiment, the first guest is an indolyl, including an indol-3-yl.

[0064] The first guest may be present in the side chain of an amino acid residue. When the first guest is phenyl, the amino acid residue may be phenylalanine.

[0065] When the first guest is a hydroxyphenyl, an example amino acid residue when the guest is 4-hydroxyphenyl is tyrosine. When the first guest is naphthyl, an example amino acid residue when the guest is naphth-2-yl is (2-naphthyl)alanine.

[0066] When the first guest is indolyl, an example amino acid residue when the guest is indol-3-yl is tryptophan. The first aryl guest may be provided within the side chain of a phenylalanine, tyrosine, indole, or naphthylalanine residue.

[0067] The second aryl guest may be selected from substituted phenyl, indolyl, such as indol-3-yl, naphthyl, such as naphth-2-yl, and anthracenyl, such as anthracen-2-yl. Wherein the aryl group is substituted with one or more substituents. The aryl group may be fully substituted. That is, each available ring position may be substituted. Wherein each substituent may be halo, preferably each substituent is fluoro.

[0068] A second aryl guest may be covalently bonded to the methylene group. The second aryl guest may be a phenyl having five halo substituents, such as five fluoro substituents, thus the second aryl guest may be a perfluorophenyl (pentafluorphenyl), which is preferably bonded to a methylene group.

[0069] The second aryl guest may be an indolyl, such as indol-3-yl, having 1 to 5 halo substituents, for example 1 to 5 fluoro substituents. The second aryl guest may be a naphthyl, such as naphth-2-yl, having 1 to 7 halo substituents, for example 1 to 7 fluoro substituents.

[0070] The second aryl guest may be anthracenyl, such as anthracen-2-yl, having 1 to 9 halo substituents, for example 1 to 9 fluoro substituents. The second aryl guest may be present in the side chain of an amino acid residue. When the second guest is perfluorophenyl, the amino acid residue may be perfluorophenylalanine.

[0071] The amino acid residues containing the second aryl guest may be provided within the polypeptide sequence having two or more consecutive amino acid residues, such as three or more, for example four or more amino acid residues, such as five or more, for example six or more amino acid residues.

[0072] The amino acid residue bearing the second aryl guest may be provided at the N-terminus of the polypeptide sequence, which may be attached to a polypeptide sequence having one or more, such as 3 or more, such as 4 or more, such as 5 or more, amino acid residues contiguous with the terminal amino acid residue.

[0073] Preferably, the polypeptide sequence has an amino acid residue at its N-terminus with a second aryl guest which is linked to one or more, such as three or more, such as four or more, such as five or more, glycine residues contiguous to the terminal amino acid residue.

[0074] The second aryl guest may be directly or indirectly covalently attached to the solid phase. The solid phase may be a polymeric phase, such as a polystyrene resin or a poly(ethylene glycol) (PEG) resin, such as Rink Amide ChemMatrix resin, where the formation of a complex binds the biomolecule to the solid phase via non-covalent interactions in the complex.

[0075] Once the biomolecule is immobilized, it can be separated from other non-insolubilized components by simple filtration. Subsequent release of the biomolecule from the complex, for example using the decomplexation methods described herein, then provides the biomolecule in a purer form.

[0076] The aryl guest may be bonded to the optionally substituted imidazolium group, for example bonded via a methylene to the 1-position of the imidazolium ring nitrogen.

[0077] The 2-, 4-, and 5-positions of the imidazolium group are preferably unsubstituted, and the 3-position of the imidazolium group is optionally substituted and may be substituted. The 3-position of the imidazolium is an alkyl group, e.g., C 1~4 It may be substituted by alkyl, such as methyl or ethyl, such as methyl.

[0078] The 3-position of the imidazolium may be bonded to a methylene group. The imidazole group may be directly or indirectly covalently attached to the solid phase via the imidazolium 3-position, for example via a methylene group attached to the imidazolium 3-position.

[0079] The amino acid residues described herein may be α-amino acid residues, β-amino acid residues, or γ-amino acid residues. The amino acid residues described herein may be α-amino acid residues.

[0080] The amino acid residues may be in the L- or D-form, typically in the L-form. In a preferred embodiment, the first guest and the second guest are both groups in the side chain of an amino acid residue. Preferably, each amino acid residue has the same stereoisomer, such as both amino acid residues having the L-form.

[0081] Biomolecules The biomolecule may be selected from a polypeptide, a polynucleotide, and a polysaccharide. The inventors have found that the ternary complex of the present invention stabilizes biomolecules such as insulin and prevents their degradation, which can be preserved without significant loss of structural or functional activity.

[0082] Biomolecules may naturally contain groups suitable for use as guests in the ternary complexes of the invention. For example, a polypeptide may contain a phenylalanine residue, the phenyl group in the side chain of which may be a suitable guest.

[0083] In other embodiments, biomolecules may be modified to include guest aryl groups such that the biomolecule can be retained in a complex. Guest groups present in the biomolecule are available for interaction with the cucurbituril host. For this reason, the first aryl guest may be provided at the end of the biomolecule, where it is likely to be freely available for interaction with the host. However, guest groups may also be provided in the mid-chain, where the guest is available for interaction. The first aryl guest is preferably provided at the end region of the biomolecule.

[0084] 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).

[0085] The polypeptide may be a protein, such as a peptide hormone or an antibody. For this reason, biomolecules are blood factors such as factors VIII and IX, Thrombolytic agents such as tissue plasminogen activator; Peptide hormones such as insulin, glucagon, growth hormone, and gonadotropins; hematopoietic growth factors such as erythropoietin and colony-stimulating factors; Interferons, such as interferon-α, -β, and -γ, Interleukin system products such as interleukin-2, Vaccines such as Hepatitis B surface antigen, Antibodies, including monoclonal antibodies, Other proteins such as tumor necrosis factor and therapeutic enzymes The polypeptide may be selected from the group consisting of:

[0086] Preferably, the biomolecule is a peptide hormone such as insulin. The polypeptide includes proteins with post-translational modifications. The polypeptide may also include unnatural amino acid residues, and the polypeptide may also include amino acid residues that are modified during post-translational modification.

[0087] A 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.

[0088] The examples herein use insulin as an exemplary polypeptide, with the bond formed to the 1-Phe residue in the B chain in insulin. Webber et al. also formed cucurbituril conjugates with the 1-Phe residue in insulin, but only in binary complexes using CB[7]. Webber et al. also demonstrated that cucurbituril conjugation with other biomolecules is possible, such as other polypeptides, including glucagon and anti-CD20 antibodies. Those skilled in the art will appreciate that the present invention may be used to form conjugates with biomolecules other than insulin.

[0089] The first guest may be provided in the N- or C-terminal region of the polypeptide, and is preferably provided in the N-terminus. Here, the terminal region may be considered as three consecutive amino acid residues in the N- or C-terminus. Typically, the first guest is present in the N- or C-terminus itself within the amino acid residue, and is preferably present in the N-terminus. The inventors have found that the binding constant for the first guest may be the largest when the first guest is present in the terminus itself with the amino acid residue, and the binding constant decreases as the first guest is provided further away from the N-terminus, such as in the second and third amino acid resides from the N-terminus.

[0090] When the biomolecule is a polypeptide, the first guest is typically present at an arylalanine residue within the polypeptide. When the biomolecule is a polypeptide, the first guest is typically present at a phenylalanine, tyrosine, indole, or naphthylalanine residue within the polypeptide.

[0091] The polynucleotide may be in a form such as DNA, RNA, or a related form such as PNA, and may be single stranded. A polynucleotide may have at least 10 contiguous nucleotides, such as at least 50, at least 100, or at least 500 nucleotides.

[0092] The first guest may be provided at the 3' or 5' terminal region of the polynucleotide. A polysaccharide may contain two or more, for example three or more, consecutive saccharide groups. The biomolecule may be a naturally occurring biomolecule or may be a variant, such as a mutant, of such a biomolecule.

[0093] Biomolecules can also be in modified form, for example containing labels for detection or capture. Typically, such functionality is provided away from the first guest so as not to interfere with the formation of the complex.

[0094] The biomolecule may be in a protected form, such as a chemically protected form. Typically, the biomolecule is not itself immobilized on the solid phase. Rather, the formation of a complex may serve to non-covalently bind the biomolecule to the solid phase through its interaction with a second guest in the complex, as shown in a preferred embodiment of the present invention, where the second guest is bound to the solid phase.

[0095] The biomolecule may be provided in an aqueous mixture, and the biomolecule may be provided together with other components, where the formation of a complex that preferentially contains the biomolecule relative to the other components may serve as a process for purifying the biomolecule from those components.

[0096] These other components may not possess suitable guest groups for complex formation, or functional groups may be present but unavailable for formation (e.g., because the guest is incorporated within another component).

[0097] Insulin The present invention is well suited for forming conjugates where the biomolecule is insulin. Insulin may be a polypeptide having an amino acid sequence corresponding to human insulin.

[0098] The insulin may be recombinantly produced insulin, thus the insulin may be human recombinant insulin. 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.

[0099] Insulin does not need to be provided in a continuously purified form. The method of the present invention can allow the formation of a complex with insulin preferentially to other compounds present. Once formed, if this complex contains a component that is bound to a solid phase, such as a second compound, it can be quickly purified from other components, for example by simple filtration.

[0100] Complex formation and decomplexation The present invention provides a method of preparing a complex of the present invention, comprising mixing a cucurbituril host together with a biomolecule having a first aryl guest and a second aryl guest, and allowing the host and guest to self-assemble.

[0101] The method may further comprise the step of at least partially purifying the complex. For example, a complex can be at least partially separated from one or more other components that are not in the complex.

[0102] The second aryl guest is preferably bound to a solid phase, such that the resulting complex is also bound to a solid phase, whereby the complex may be at least partially purified by recovering the solid phase, for example by filtration, to separate the complex from other components.

[0103] The invention also provides a method of decomplexing a complex of the invention, comprising treating the complex with an external stimulus, and allowing the external stimulus to displace at least a first aryl guest from the complex.

[0104] Release of biomolecules may be achieved when needed and may be considered as release on demand. The external stimulus may be the addition of a competing guest, which may be or include adamantanamine (ADA) or memantine (DMADA).

[0105] The formation and decomplexation may be carried out under aqueous conditions. Adventurously, the formation and decomplexation steps may also be carried out at ambient temperature, for example at a temperature in the range of 5-30°C.

[0106] Conjugation and deconjugation may be carried out at temperatures that do not involve degradation of the biomolecule, and the integrity of the biomolecule may be maintained during the conjugation and deconjugation process. Stabilization of biomolecules The complexes of the present invention may be used to stabilize biomolecules for storage, which can be easily and quickly released from the complex when required, thus limiting the degradation of the biomolecule and maintaining its activity upon release.

[0107] Here, the complex of the invention is preferably bound to said solid phase, for example the second guest is bound to a solid phase, such as a polymer phase. Thus, there is provided the use of the conjugates to stabilise biomolecules such as proteins.

[0108] The stability of a biomolecule may be assessed by structural analysis such as circular dichroism, which may include comparison of the secondary and tertiary structure of the biomolecule before and after complexation. The stability of a biomolecule may be assessed by suitable activity assays, which may include cell-based studies.

[0109] Biomolecule purification The selective capture of a biomolecule in a complex also allows the biomolecule to be separated from other components in a mixture, thereby at least partially purifying the biomolecule, for example, the complex can be bound to a solid phase to allow recovery of the complex by simple filtration and separation from other solubilized components of the mixture.

[0110] Thus, there is provided a method for at least partially purifying a biomolecule from a mixture comprising the steps of providing a biomolecule in a mixture, such as an aqueous mixture, where the biomolecule has a first aryl guest, providing a cucurbituril host and a second aryl guest in the mixture, allowing the host and guest to self-assemble, and subsequently separating the resulting complex from the mixture.

[0111] experiment Cucurbit[n]uril (CB[n]) macrocyclic hosts are ideal for conjugating peptides due to their high binding affinity and range of guests. 6~11 Urbach and coworkers showed that a homopeptide dimer between two FGG tripeptides and CB[8] has a high binding avidity (K ca. 10 11 M -2 ) was reported. 6 This homodimer is a supramolecular oligomer. 12 ,polymer 13、14 , hydrogel 15、16 , and protein / peptide organization 17 They have been employed as versatile building blocks in the design and fabrication of peptides. Although significant progress has been made, major challenges remain, such as the desired quantitative formation of heteropeptide dimers without homodimerization.

[0112] Herein, we employ CB[8] to mediate the heterodimerization of canonical aromatic peptides and noncanonical L-perfluorophenylalanine (F')-containing peptides (Figure 1a). Recently, we reported that a first guest, an electron-deficient perfluorophenyl, and a second guest, an electron-rich phenyl, can exclusively form CB[8]-mediated heteroternary complexes via host-enhanced polar-π interactions. 18、19 For this reason, we hypothesized that F'-containing peptides (F'GG) would form 1:1 complexes with CB[8] exclusively, and homodimerization would be avoided by electrostatic repulsion within the 2:1 complex. Subsequent association of various aromatic peptides (e.g., WGG) with the F'GG-CB[8] complex may enable the access of novel host-enhanced heteropeptide dimers with superior binding avidity (Figure 1a).

[0113] Five model aromatic tripeptides containing L-perfluorophenylalanine (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 1a-b). An equimolar mixture of F'GG, CB[8], and XGG would result in exclusive heterodimers instead of the equilibrium mixture containing homodimers. Two additional series of tripeptides (GGX, GXG) containing aromatic amino acids at either the C-terminus or mid-chain were synthesized to explore the effect of various second guests and their position in the oligopeptide (shown below). After elucidation of the binding thermodynamics, we applied this heterodimerization to the on-resin recognition and isolation of aromatic tripeptides from peptide mixtures. 21、22 was achieved, demonstrating high efficiency and selectivity (Figure 1c).

[0114] [ka]

[0115] [ka]

[0116] 1 H and 19 NMR titrations of F were performed to probe the heteropeptide dimerization in CB[8] (Figure 2a) (additional data not shown). Titration of WGG into a 1:1 mixture of F'GG-CB[8] led to the progressive appearance of indole protons at 6.25–7.10 ppm. Due to shielding from the CB[8] cavity, these proton peaks showed an upfield shift compared to free WGG, suggesting that the indole groups in F'GG-CB[8]-WGG are located in a different chemical environment than unbound WGG. This titration 19 The results were also monitored by F NMR (Fig. 2a). The fluorine peaks of the new groups gradually appeared while the peaks of F'GG-CB[8] disappeared. In addition, an equal mixture of F'GG, CB[8], and XGG was characterized by high-resolution ESI-MS (Fig. 2b). All ion peaks for 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 confirmed the successful formation of the novel heteropeptide dimers.

[0117] Isothermal titration calorimetry (ITC) was used to study the binding thermodynamics of heteropeptide dimerization (Fig. 2c and additional data not shown). Titration of F'GG (3.0 mM) into CB[8] (0.1 mM) resulted in a stepwise binding curve with two changes in the molar ratio at 1.0 and 2.0 (Fig. 2c). The first-step binding constant (K1) was 6.6 × 10 5 M -1 However, the second stage (K2) is 9.4×10 3 M -1 This means that K1>K2, which indicates negative cooperativity. 18、19、23Such a favorable 1:1 conjugation allows for secondary utilization of electron-rich aromatic peptides. Titration of XGG (X = W, F, Y, Np; 3.0 mM) into F'GG-CB[8] (0.2 mM) resulted in four binding curves with distinct shifts at the molar ratio of 1.0 (Figure 2d), indicating successful incorporation of XGG into F'GG-CB[8].

[0118] Table 1 shows that all XGG peptides have high binding affinity (K a >10 4 M -1 ), which highlights the thermodynamic stability of heteropeptide dimerization. YGG (Fig. 2d, green) exhibits a relatively low K because its para-substituted hydroxyl group may reduce the enthalpic contribution and weaken the second association. a showed 24 Nevertheless, the overall binding constant (KK a ) are all 10 10 M -2 This is because the parent dimer (e.g., F'GG-WGG 25、26 , K dimer 1M -1 ) shows a significant enhancement compared to F'GG-CB[8]-FGG (heterodimer 2.4 × 10 11 M -2 ) General K1K a Due to its enhanced polar interactions, 2FGG-CB[8] (homodimer, 1.5 × 10 11 M -2 ) higher 6 Notably, no secondary association was observed with the nonaromatic analogues (KGG, EGG, LGG), highlighting the selectivity for aromatic over nonaromatic peptides.

[0119] To understand the effect of the aromatic position on heterodimerization, we investigated the K-relationship between GXG and GGX (X = W, F, Y, Np) with F'GG-CB[8]. a was determined by ITC. The shift of aromatic residues from the N-terminus to the C-terminus was aThis resulted in a notable decrease in the binding affinity (Table 1). The extended distance between the positive charge at the N-terminus and its aromatic motif weakens the ion-dipole interactions at the entrance of CB[8], reducing the secondary binding affinity. This is demonstrated by GGY, which shows no secondary binding to F'GG-CB[8]. Seven novel derivatized heteropeptide dimers, F'GG-CB[8]-GXG, and F'GG-CB[8]-GGX, broaden the scope of host-enhanced heteropeptide dimerization. The demonstrated binding selectivity to aromatic residues is an advantage of this system, allowing the utilization of a variety of peptides and proteins. Compared to previous reports on CB[8]-peptide heteroternary complexes, 27 However, the system described herein is simply based on the F' amino acid, which is readily accessible to ligation reactions in chemical biology and biochemistry.

[0120] [Table 1]

[0121] The thermodynamic mechanism behind peptide heterodimerization is attributed to social self-sorting (Fig. 2e), which is consistent with previous reports. 18、19 Two pathways exist in equimolar mixtures of F'GG, CB[8], and XGG: social self-sorting (heterodimerization) and narcissistic self-sorting (homodimerization). When there is a ratio of hetero- to homodimer abundance (e.g., WGG, R = 2291) compared to homodimerization (Table S1), social self-sorting is favored, confirming quantitative heterodimerization in the complex mixture. Notably, simply mixing two aromatic tripeptides with CB[8] does not result in quantitative heterodimerization (data not shown).

[0122] After elucidation of the thermodynamics, we demonstrated the utility of heteropeptide dimers to achieve on-resin recognition of aromatic peptides (Figure 1c). Using confocal fluorescence imaging, we probed the interfacial recognition via a fluorescently tagged peptide (WGGGGG-dansyl). 9 (Figure 3a-b). A buffer 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) by vigorous shaking for 10 min at 25 °C. Upon laser irradiation (λ = 405 nm), confocal fluorescence images were captured under the grey area. Figure 3c-f shows that the resin with F'GGGGG-CB[8]-WGGGGG-dansyl exhibited significantly higher fluorescence than F'GGGGG, F'GGGGG-CB[8]-WGG, and F'GGGGG-WGGGGG-dansyl (data not shown), suggesting successful heteropeptide dimerization on the resin.

[0123] UV experiments were performed to test the adsorption efficiency via quantification of aromatic peptides present before and after on-resin treatment (Figure 4a). A typical experiment entailed mixing WGG-CB[8] (1.0 mM) with F'GGGGG-resin (10.0 mM) for 10 min at 25 °C. The adsorption strength of resin-treated WGG-CB[8] (gray) showed a decrease compared to untreated (purple) (Figure 4b). The adsorption efficiency for on-resin recognition was 77%, while the recognition of free WGG by physical adsorption was only 19% (data not shown). The adsorbed WGG was released and reused via competitive binding with memantine hydrochloride (DMADA), thereby regenerating the resin (additional data not shown).

[0124] Multiple cycles of on-resin recognition were performed to evaluate reusability (Figure 4c) (additional data not shown). The recognition-regeneration experiment for WGG-CB[8] was repeated for three cycles using the same batch of resin. The on-resin regeneration efficiency remained above 98% over multiple cycles with complete release of WGG without any residue accumulation (Figure 4c). This confirms the regeneration of F'GGGGG-functionalized resin, endowing the whole process with high sustainability for practical use. We further explored the selective isolation of aromatic peptides via a three-cycle recognition-release experiment using a peptide mixture of WGG, KGG, EGG, and LGG ([XGG] = 1.0 mM) in the presence of 1.0 mM CB[8] (Figure 4d, and the following related discussion along with Table 4 and Figure 8). The ratio of WGG (purple) in the mixture is increased from 26 to above 95% after three cycles (Figure 4d). In addition, residual DMADA and DMADACB[8] can be removed via liquid-phase chromatography. This facile strategy to obtain highly pure aromatic peptides via on-resin heteropeptide dimerization is readily amenable to automation.

[0125] To broaden the applicability of this approach, we exploited the interfacial recognition for insulin stabilization and its on-demand release from the resin (Figure 4e and Table 2) (see below). Insulin is a widely used biopharmaceutical for the treatment of diabetes. 28 Its limited stability requires strict storage conditions (e.g., 2-6°C) due to its tendency to form immunogenic fibrillar aggregates in solution. 29 Insulin has an N-terminal phenylalanine, which can serve as a guest.

[0126] UV experiments quantified insulin absorbance and on-demand release onto the resin. Insulin adsorption efficiency was calculated to be 94%, and its adsorption strength decreased after treatment with F'GGGGG-CB[8] resin (Fig. 4f and Fig. 6) (see also Fig. 5 with UV spectrum for insulin on resin). Through competitive binding, insulin was transferred by DMADA with 95% regeneration efficiency (Fig. 4g) over 8 days of storage at room temperature, indicating long-term stability (Fig. 7 and Table 3, as shown below). Our approach provides a route to store insulin under ambient conditions, eliminating the current need for refrigeration.

[0127] In conclusion, we have introduced a new type of quantitative heteropeptide dimerization. Through host-enhanced polar interactions, the binding affinity between aryl and perfluorophenyl groups from two different peptides can be determined by the K a Up to 10 5 M -1 , K1K a Up to 10 11 M -2 , confirming the exclusive formation of heteropeptide dimers. To demonstrate utility, the solution-phase host-guest complex (F'GG-CB[8]-XGG) was transferred to a solid-liquid interface where, in addition to on-resin recognition and isolation of aromatic peptides, insulin stabilization and on-demand release were achieved under ambient conditions. This comprehensive approach allows for the accumulation and isolation of aromatic-rich biopolymers useful in biomedical research. We anticipate that this work will advance research into the exploitation of heteropeptide dimerization as a versatile strategy for a wide range of life science applications.

[0128] Materials and Instrumentation material 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-O H, diisopropylethylamine (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), disodium hydrogen phosphate (Na2HPO4), deuterium oxide (DO, D, 99.8%), 1,3-dimethylaminoadamantane hydrochloride (memantine hydrochloride, DMADA), Rink Amide-AM resin, H-Rink Amide ChemMatrix resin (surface loading of functionalization approximately 0.5 mmol / g), insulin (human recombinant). Phosphate buffer was prepared by adding 10 mM NaH2PO4 solution to 10 mM Na2HPO4 solution until the pH was equal to 7.0. Cucurbit[8]uril (CB[8]) was synthesized as previously reported (see Kim) and purified to adjust the molecular weight of CB[8] to 1600 g / mol. Water was obtained from a Milli-Q Integral water purification system (22.5 MΩ-cm).

[0129] Peptide synthesis and purification All tripeptides (XGG, GXG, GGX) were synthesized on an automated microwave peptide synthesizer (Liberty Blue, CEM) using standard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. The peptides were cleaved from the Rink Amide-AM resin using a mixture of 95% TFA, 2.5% TIPS, and 2.5% HO and shaken at room temperature for 1 h. The resin was filtered and washed with the cleavage mixture. After evaporating off most of the cleavage mixture, the peptides were ground and washed three times with chilled diethyl ether. The crude peptides were purified using reversed-phase high-performance liquid chromatography (RP-HPLC) using a Phenomenex C18 Kinetic-Evo column with a pore size of 5 μm, particle size of 110 Å, and dimensions of 150 × 21.2 mm. The water mobile phase contained 0.1% TFA. The purification gradient was set up from 5% acetonitrile and 95% water to 100% acetonitrile. The dried peptide was obtained by lyophilization (as TFA salt) and then confirmed by NMR and ESI-MS. Using the same method as above, peptide F'GGGGG on resin was synthesized using H-Rink Amide ChemMatrix resin. This F'GGGGG functionalized resin was washed with DMF three times and then placed in D2O / H2O (fresh D2O / H2O was replaced every day for three days). The washed F'GGGGG resin was then stored in D2O / H2O in a refrigerator at 3.85°C (277K).

[0130] Nuclear magnetic resonance spectroscopy (NMR) Pure peptides 1 H, 13 C, and 19 F NMR spectra were obtained in DO at 24.85 °C (298 K) on a Bruker AVANCE400 (400 MHz) instrument equipped with a TCI Cryoprobe (500 MHz) and a Bruker AVANCE500 instrument. 1 H and 19F NMR spectra were obtained on a Bruker AVANCE400 (400 MHz) instrument in phosphate buffer (D2O) at pH 7.0. Chemical shifts were referenced to the residual solvent peak of HDO at 4.79 ppm.

[0131] Isothermal titration calorimeter (ITC) ITC experiments were run on a Malvern MicroCal Auto-ITC200 instrument at 24.85 °C (298 K) in 10 mM phosphate buffer, pH 7.0, with a single injection of 0.6 μL and 32 consecutive injections of 1.2 μL with an injection interval of 90 s. The first data point was removed before analysis. ITC data were fitted by MicroCal Analysis Centre software using the one set of sites model. In a typical experiment for homoternary complexation, the host molecule (CB[8]) was loaded into the sample cell at a concentration of 0.1 mM and the guest peptide was loaded into the syringe at a concentration of 2.0 mM. Injection and interval settings were the same as above. The first data point was removed from the data set before analysis. The resulting ITC curves were fitted by MicroCal Analysis Centre software using the sequential binding model. All parameters were averaged over three replicates (standard deviation n = 3). K a Value < 10 2 M -1 was too low to be detected.

[0132] Electrospray ionization mass spectrometry (ESI-MS) ESI-MS data were obtained on a Thermo Fisher Q Exactive Orbitrap mass spectrometry equipped with a nanospray ion source. In all experiments, the positive mode was selected with a working temperature of 320 °C and a capillary voltage of 1.5 kV. All sample solutions were prepared in pure water.

[0133] Ultraviolet (UV) spectroscopy UV spectra were obtained on a Cary4000 UV-Vis spectrometer. A pair of quartz cuvettes with a 2 mm gap was used. Double beam mode with a fixed spectral bandwidth of 2 nm and baseline correction was selected. In a typical run, 300 μL of 1 mM solution was transferred to the quartz cuvette and scanned against phosphate buffer in D2O. For better quantification, UV spectra were obtained in the linear absorption range (0.2-0.8).

[0134] Confocal microscopy Imaging on the resin was performed with a Leica Stellaris5 confocal microscope (Leica Microsystems) using a 40x oil immersion objective and a numerical aperture of 1.3. A suspension of resin beads was dropped onto a cover slip and a stack of 50 images was acquired with a resolution of 1024x1024 pixels at _EXC405nm. The stack border was chosen at a point where there was no discernible difference in signal to noise at either end (edge) of the bead. The 3D dataset was processed with Fiji / ImageJ and maximum intensity Z-projections were obtained to compare the mean fluorescence intensity across the resin bead. Three groups of resin beads were tested: Group A, untreated 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]), Resin-G5F' with WGG, CB[8], then DMADA solution (Resin-G5F'+WGG+CB [8]+DMAMA), C group, 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). The three groups of resin beads were prepared according to the procedure in UV spectroscopy above. Then, all samples were drained and the same amount of pH 7.0 phosphate buffer was added to obtain the suspension (10 mM) for imaging.

[0135] Circular dichroism (CD) spectroscopy CD measurements were performed on a Chirascan spectrometer (Applied Photophysics). Scans were taken from 200 to 500 nm at 1.0 nm intervals at 298 K. Acquisition time in a quartz cuvette with a path length of 0.1 cm was 0.5 s. CD spectra were collected using solutions from the UV run of the 8-day storage and release experiment described above. Untreated insulin solutions were 0.2 mM. All solutions were diluted 2-fold before measurements. All scans were collected and averaged over three scans with background noise correction.

[0136] Procedure for on-resin recognition The standard procedure for NMR quantification for on-resin recognition within the peptide mixture is shown as follows: A solution of mixed peptides containing WGG, KGG, LGG, EGG, CB[8] ([XGG] = CB[8] = 1 mM) was used as the raw cycle 0 solution. In cycle 1, 1.0 ml of mixed peptides (1 mM) was added to the 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 post-release solution. In cycle 2, fresh CB[8] (2.88 mg, 1.8 μmol) was added to 0.9 ml of the post-release solution from cycle 1 (2 mM DMADA, 1.8 μmol) to combine with the excess DMADA and isolated WGG. Approximately 0.1 ml of solution was lost after each cycle. To this new solution, resin was added, shaken for 45 minutes, drained, and then 0.9 ml of 2 mM DMADA solution was added to generate the post-release solution from cycle 2. In cycle 3, fresh CB[8] (2.56 mg, 1.6 μmol) was added to 0.8 ml of the post-release solution from cycle 2 (2 mM DMADA, 1.6 μmol). To this new solution, resin was added, shaken for 45 minutes, drained, and then 0.8 ml of DMADA solution was added to generate the post-release solution from cycle 3. The untreated cycle 0 solution and the subsequently released solutions from each cycle were examined by NMR. After NMR analysis, the relative amount of WGG was determined by integrating a set of doublet peaks at 3.36 ppm. The relative amount of KGG was determined by integrating a set of triplet peaks at 3.02 ppm. The relative amount of EGG was determined by integrating a set of triplet peaks at 2.37 ppm. There were overlapping singlet peaks of KGG, LGG, and EGG at 3.95 ppm, so the relative amount of LGG was determined by subtracting the relative amounts of KGG and EGG from the integral of the peak at 3.95 ppm.

[0137] The standard procedure of UV quantification for on-resin recognition of peptides is shown as follows. Using the model tripeptide WGG as an example, a resin DO suspension of 10 μmol F'GGGGG equivalents was placed in a 12 ml syringe equipped with a polyester frit (r=8.0 mm, h=2.0 mm). After draining the DO, 1 ml of 1 mM WGG:CB[8]=1:1 solution (untreated solution in DO, phosphate buffer, pH=7.0) was added to the resin. Then, a plunge and a stopper were attached to the syringe to form a closed chamber. The syringe was transferred to a wrist-action shaker and shaken for 10 min. Then, the liquid in the syringe (resin treatment solution) was completely drained and the resin was washed once with DO. After that, 1 ml of 2 mM DMADA solution (in DO, phosphate buffer, pH=7.0) was added to the resin and shaken for another 45 min. The solution was drained for further testing (post-release solution). For the efficiency test over three cycles (Figure 4c), the 1:1 WGG:CB[8] solution and the DMADA solution were alternately added to the resin, shaken, and drained for three cycles to generate a series of resin treatment and post-release solutions.

[0138] The standard procedure of UV quantification for on-resin recognition of insulin is shown as follows: Insulin (human recombinant) was suspended at 0.2 mM using 0.22 μm filtered H2O. 1N HCl was immediately added to a 5 mM acid concentration. The solution was gently stirred until completely dissolved, filtered through a 0.22 μm filter, and stored at 3,85 °C (277 K). The method was slightly modified from the Cold Spring Harbor Protocol (see recipe: insulin). The insulin solution was warmed to room temperature before being used for on-resin recognition. In a typical experiment, a resin H2O suspension of 8 μmol F'GGGGG equivalents was placed in a 12 ml syringe equipped with a polyester frit (r = 8.0 mm, h = 2.0 mm). After draining the H2O, 8 ml of 0.1 mM CB[8] solution was added to the resin, and a plunge and stopper were attached to the syringe to form a closed chamber. The syringe was transferred to a wrist action shaker and shaken for 12 hours. The CB[8] solution was then drained, after which the resin was washed once with H2O, and 0.8 ml of 0.2 mM insulin solution was added to the resin and gently shaken for 3 hours. The liquid in the syringe (resin treatment solution) was then completely drained, and the resin was washed once with H2O. Then, 0.8 ml of 2 mM DMADA solution (in 5 mM HCl) was added to the resin and gently shaken for another 3 hours. The solution (post-release solution) was drained. The untreated insulin solution, resin treatment solution, and post-release solution were immediately measured by UV spectroscopy.

[0139] The 8-day storage and release experiment was carried out as follows: 9 syringes were prepared with 8 μmol F'GGGGG equivalents of resin in HO suspension each. On day 0, all 9 syringes were drained, conjugated with CB[8], washed, conjugated with insulin, drained (to obtain resin treatment solution), and washed as per the standard procedure for UV quantification for on-resin recognition of insulin described above. All syringes with resin were kept at room temperature. Starting from day 0 to day 8, 0.8 ml of 2 mM DMADA solution was added to the syringe with resin every day (once a day to obtain post-release solution). All the obtained solutions were immediately analyzed by UV spectroscopy. The numbers to calculate the released percentage in Figure 4g were absorbance at 276 nm.

[0140] Synthesis and characterization of model peptides (S)-2-amino-N-(2-((2-amino-2-oxoethyl)amino)-2-oxoethyl)-3-(perfluorophenyl)propenamide (F'GG) 1 H NMR (500 MHz, D2O, ppm): δ = 4.32 (t, 1H), 3.99 (q, 2H), 3.93 (s, 2H), 3.37 (d, 2H); 13 C NMR (125 MHz, 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, DO, ppm): δ = -142.65, -154.41, -162.18; ESI MS for [MH]: calculated m / z = 369.10, found m / z = 369.17.

[0141] (S)-N-(2-amino-2-oxoethyl)-2-(2-aminoacetamido)-3-(perfluorophenyl)propanamide (GF'G) 1H NMR (500 MHz, 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); 13 C NMR (125 MHz, 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 F NMR (375 MHz, DO, ppm): δ = -143.48, -155.99, -162.89; ESI MS for [MH]: calculated m / z = 369.10, found m / z = 369.17.

[0142] (S)-2-(2-(2-aminoacetamido)acetamido)-3-(perfluorophenyl)propanamide (GGF') 1 H NMR (500 MHz, 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 (125 MHz, 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, DO, ppm): δ = -143.47, -156.31, -163.04; ESI MS for [MH]: calculated m / z = 369.10, found m / z = 369.17.

[0143] (S)-2-amino-N-(2-((2-amino-2-oxoethyl)amino)-2-oxoethyl)-3-(1H-indol-3-yl)propanamide (WGG) 1 H NMR (500 MHz, 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); 13 C NMR (125 MHz, DO, 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; ESI MS for [MH]: calculated m / z = 318.16, found m / z = 318.34.

[0144] (S)-N-(2-amino-2-oxoethyl)-2-(2-aminoacetamido)-3-(1H-indol-3-yl)propanamide (GWG) 1 H NMR (500 MHz, 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, DO, 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; ESI MS for [MH]: calculated m / z = 318.16, found m / z = 318.34.

[0145] (S)-2-(2-(2-aminoacetamido)acetamido)-3-(1H-indol-2-yl)propanamide (GGW) 1 H NMR (500 MHz, 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 (125 MHz, DO, ppm): δ = 176.11, 170.80, 167.56, 136.05, 126.84, 124.49, 121.89, 119.30, 118.29, 111.84, 108.86, 54.13, 42.11, 40.23, 26.96; ESI MS for [MH]: calculated m / z = 318.16, found m / z = 318.34.

[0146] (S)-2-amino-N-(2-((2-amino-2-oxoethyl)amino)-2-oxoethyl)-3-phenylpropanamide (FGG) 1 H NMR (500 MHz, 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, DO, ppm): _ = 173.97, 171.27, 169.82, 133.68, 129.35, 129.14, 128.00, 54.41, 42.26, 41.90, 36.69; ESI MS for [MH]+: calculated m / z = 279.15, found m / z = 279.34.

[0147] (S)-N-(2-amino-2-oxoethyl)-2-(2-aminoacetamido)-3-phenylpropanamide (GFG) 1 H NMR (500 MHz, 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, DO, ppm): δ = 173.76, 173.49, 166.96, 136.05, 129.14, 128.80, 127.28, 55.32, 41.99, 40.18, 36.93; ESI MS for [MH]: calculated m / z = 279.15, found m / z = 279.34.

[0148] (S)-2-(2-(2-aminoacetamido)acetamido)-3-phenylpropanamide (GGF) 1 H NMR (500 MHz, 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, DO, ppm): δ = 175.70, 170.82, 167.57, 136.41, 129.14, 128.69, 127.11, 54.68, 42.01, 40.29, 36.98; ESI MS for [MH]: calculated m / z = 279.15, found m / z = 279.34.

[0149] (S)-2-(2-(2-aminoacetamido)acetamido)-3-(4-hydroxyphenyl)propanamide (GGY) 1H NMR (500 MHz, 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, DO, ppm): δ = 175.77, 170.79, 167.57, 154.37, 130.49, 128.22, 115.38, 54.84, 42.01, 40.30, 36.18; ESI MS for [MH]: calculated m / z = 295.14, found m / z = 295.34.

[0150] (S)-2-amino-N-(2-((2-amino-2-oxoethyl)amino)-2-oxoethyl)-3-(naphthalen-2-yl)propanamide (NpGG) 1 H NMR (500 MHz, 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); 13 C NMR (125 MHz, DO, 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; ESI MS for [MH]: calculated m / z = 329.16, found m / z = 329.34.

[0151] (S)-N-(2-amino-2-oxoethyl)-2-(2-aminoacetamido)-3-(naphthalen-2-yl)propanamide (GNpG) 1 H NMR (500 MHz, 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, DO, 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; ESI MS for [MH]: calculated m / z = 329.16, found m / z = 329.34.

[0152] (S)-2-(2-(2-aminoacetamido)acetamido)-3-(naphthalen-2-yl)propanamide (GGNp) 1 H NMR (500 MHz, 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); 13 C NMR (125 MHz, DO, 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; ESI MS for [MH]: calculated m / z = 329.16, found m / z = 329.34.

[0153] (S)-2,6-Diamino-N-(2-((2-amino-2-oxoethyl)amino)-2-oxoethyl)hexanamide (KGG) 1 H NMR (500 MHz, 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, DO, ppm): δ = 05, 25.74; ESI MS for [MH]: calculated m / z = 261.12, found m / z = 261.34.

[0154] (S)-2-Amino-N-(14-amino-2,5,8,11,14-pentaoxo-3,6,9,12-tetraazatetradecyl)-3-(perfluorophenyl)propenamide (F'GGGGG) 1 H NMR (700 MHz, D2O, ppm): δ =4.30 (t, 1H), 4.02 (m, 8H), 3.95 (s, 2H), 3.37 (d, 2H); 19 F NMR (375 MHz, DO, ppm): δ = -142.63, -154.54, -162.22; ESI MS for [MH]: calculated m / z = 540.16, found m / z = 540.67.

[0155] 2-Amino-N-(14-amino-2,5,8,11,14-pentaoxo-3,6,9,12-tetraazatetradecyl)acetamide (GGGGGG) 1 H NMR (700 MHz, DO, ppm): δ = 4.09 (s, 2H), 4.04 (d, 4H), 4.02 (s, 2H), 3.95 (s, 2H), 3.93 (s, 2H); ESI MS for [MH]: calculated m / z = 360.16, found m / z = 360.50.

[0156] (S)-2-Amino-N-(17-((5-(dimethylamino)naphthalene)-1-sulfonamido)-2,5,8,11,14-pentaoxo-3,6,9,12,15-pentaazaheptadecyl)-3-(1H-indol-3-yl)propenamide (WGGGGG-dansyl) 1 H NMR (700 MHz, D2O, ppm): δ = 8.68 (d, 1H), 8.47 (d, 1H), 8.36 (d, 1H), 7.97 - 7.85 (m, 3H), 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); ESI MS for [MH]+: calculated m / z = 765.31, observed m / z = 765.34.

[0157] Insulin recognition on resin As shown in Fig. 5a, under the same experimental conditions as Fig. 4f, insulin showed minimal adsorption to F'GGGGG-resin without CB[8]. In Fig. 5b, c, by using the same ChemMatrix resin but loaded with a different peptide, GGGGGG, other than F'GGGGG, insulin showed negligible adsorption in both cases, regardless of the use of CB[8]. In Fig. 5d, there was almost no adsorption to the blank resin (purchased H-Rink Amide ChemMatrix resin without any loaded peptide), even when insulin and CB[8] were used simultaneously. This confirmed that the adsorption of insulin-CB[8] to F'GGGGG-resin was achieved by host-enhanced polar-π interactions.

[0158] A higher efficiency of 94% is observed when F'GGGGG resin is treated with insulin (0.2 mM) solution compared to 77% when F'GGGGG resin is treated with WGG (1.0 mM) solution. This phenomenon is indeed one of the advantages for insulin stabilization on the resin. The K of WGG and FGG a Considering that the values ​​are very close, the difference in adsorption efficiency between WGG and insulin is not simply related to equilibrium calculations. Unlike WGG, insulin has two relatively long peptide chains consisting of 51 amino acids, which allows its relatively large size to kinetically trap insulin in the network structure of cross-linked PEG (the material of ChemMatrix resin). This can increase the adsorption efficiency to about 94%, which is significantly higher than that of WGG, a small free tripeptide.

[0159] [Table 2]

[0160]

number

[0161] Verification of the secondary structure of insulin after release As shown in Figure 7 and Table 3, all three samples showed highly similar CD curves when released back into the liquid phase after 2, 5, and 8 days of storage on the functionalized resin compared to the freshly prepared insulin solution, which indicated that the secondary structure of insulin remained unchanged after 2, 5, and 8 days of storage at room temperature.

[0162] [Table 3]

[0163] Additional Experiments Selective isolation of aromatic peptides on resin The released DMADA-CB[8] complex may have some weak interactions (i.e., electrostatic interactions, hydrogen bonds) with the remaining tripeptide, which may result in some changes in chemical shifts after release. The yield of selectively separated WGG for a single cycle was 77% (data not shown), and thus the final yield of WGG after three cycles of selective separation was 77% × 77% × 77% = 45.6% (see also Figure 8).

[0164] [Table 4]

[0165] References

[0166] [Table 5]

Claims

1. A ternary complex of a cucurbit uryl host having a first aryl guest and a second aryl guest, wherein the first aryl guest is a biomolecular group, the first aryl guest and the second aryl guest are different, and at least one aryl guest is fluorinated.

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

3. The complex according to claim 2, wherein the cucurbit uryl host is CB[8].

4. The composite according to claim 1, wherein the second aryl guest is a fluoroaryl group.

5. The complex according to claim 4, wherein the first aryl guest is selected from phenyl, naphthyl such as naphtho-2-yl, hydroxyphenyl such as 4-hydroxyphenyl, and indolyl such as indole-3-yl.

6. The composite according to claim 4, wherein the fluoroaryl is fluorophenyl.

7. The composite according to claim 6, wherein the fluorophenyl is perfluorophenyl.

8. The composite according to claim 1, wherein the second aryl guest is covalently bonded directly or indirectly to the solid phase.

9. The complex according to claim 1, wherein the biomolecule is selected from the group consisting of polypeptides, polynucleotides, and polysaccharides.

10. The complex according to claim 1, wherein the biomolecule is a polypeptide.

11. The complex according to claim 10, wherein the polypeptide is a protein.

12. The complex according to claim 10, wherein the polypeptide is a peptide hormone or an antibody.

13. The complex according to claim 1, wherein the first aryl guest is provided within the side chain of a phenylalanine, tyrosine, tryptophan, or naphthylalanine residue.

14. The complex according to claim 1, wherein the biomolecule is insulin.

15. The complex according to claim 14, wherein the insulin is human insulin, or optionally recombinant human insulin.

16. The complex according to claim 14, wherein the first guest is a phenyl group within the phenylalanine residue at position 1 of insulin.

17. A method for decompositing a complex according to any one of claims 1 to 16, comprising the steps of: treating the complex with a competing guest; and enabling the competing guest to move at least a first aryl guest from the complex.

18. The method according to claim 17, wherein the competing guest is adamanthanamine (ADA) or memantine (DMADA), or comprises adamanthanamine (ADA) or memantine (DMADA).

19. A method for forming a complex according to any one of claims 1 to 16, comprising the steps of: mixing a cucurbit uryl host with a biomolecule having a first aryl guest and a second aryl guest, wherein the first and second aryl guests are different and at least one aryl guest is fluorinated; and enabling the host and guests to self-assemble.

20. The method according to claim 19, further comprising the step of purifying the complex at least partially, such as by filtration.

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