Photoswitchable compounds and affinity ligands and their use for optical control of affinity matrices

Abstract

The present disclosure relates to photoswitchable azobiaryl compounds, photoswitchable affinity ligands, photoswitchable affinity matrices, the use of photoswitchable compounds, photoswitchable affinity ligands, or photoswitchable affinity matrices for isolating and / or purifying target molecules, methods for isolating and / or purifying target molecules, and processes for the preparation of photoswitchable azobiaryl compounds. JPEG2026507223000054.jpg3994

Description

[Technical Field]

[0001] The present disclosure relates to photoswitchable azobiaryl compounds, photoswitchable affinity ligands, photoswitchable affinity matrices, the use of photoswitchable compounds, photoswitchable affinity ligands, or photoswitchable affinity matrices for isolating and / or purifying target molecules, methods for isolating and / or purifying target molecules, and processes for the preparation of photoswitchable azobiaryl compounds. [Background technology]

[0002] Generally, chromatographic methods are used to separate and / or purify molecules, compounds, or substances of interest, such as proteins, nucleic acids, virus particles, cells, and polysaccharides, from compositions of different substances. Affinity chromatography specifically involves passing such a composition over an affinity matrix containing a ligand (i.e., a specific binding partner) that is specific for the target molecule contained in the composition.

[0003] Upon contact with the ligand under conditions that allow strong binding, the target molecule binds to the matrix and is therefore retained from the composition. After subsequent depletion of contaminating components, an elution buffer is typically used to favor the release of the target molecule from the affinity matrix in a final step.

[0004] Affinity chromatography offers certain advantages over other types of chromatography: for example, it provides a purification method that can isolate a target protein from a mixture of the target protein and other biomolecules in a single step with high selectivity and high yield.

[0005] Despite the advantages of current affinity chromatography methods, there is a need to improve the mechanism of elution, which is often seen as the most critical process step. Elution should ideally be performed in a manner that keeps the affinity matrix intact, allowing for regeneration and multiple uses.

[0006] Binding of target molecules to affinity matrices occurs under mild buffer conditions that mimic their natural environment with respect to pH and ionic strength, while the elution step often requires dramatic changes, for example, by strongly altering pH, polarity, or ionic strength (Hage DS et al., J Pharm Biomed Anal. 2012 Oct;69:93-105. doi:10.1016 / j.jpba.2012.01.004.).

[0007] Nonspecific elution conditions, such as altered pH, high concentrations of salt, organic cosolvents, detergents, metal ions, chelating agents, or reducing agents, often damage the target molecule and / or the affinity matrix. Particularly when the target molecule is a protein, such elution conditions can result in denaturation, aggregation, or chemical modification, thus interfering with its physicochemical properties or functional activity.

[0008] Alternatively, a competitor, e.g., imidazole in the case of the His6 tag, can be added to the elution buffer to displace the target molecule from binding to the affinity ligand (Hochuli, E. et al., Nat Biotechnol 6, 1321-1325 (1988). https: / / doi.org / 10.1038 / nbt1188-1321). Obviously, using such small molecules as competitors for elution leads to contamination of the solution containing the purified target molecule. Consequently, these reagents must be removed in additional, time-consuming purification steps, e.g., by dialysis or gel filtration. Furthermore, after elution of the target molecule, the affinity matrix must be regenerated before the next sample application, a resource-consuming procedure.

[0009] Perhaps the greatest success of large-scale affinity chromatography has been achieved in the field of biopharmaceutical monoclonal antibody purification. In modern biotechnology and medicine, antibodies are an essential part of nearly every application area. In particular, antibodies have been a consistent game-changer for the treatment of life-threatening diseases such as cancer or autoimmune disorders.

[0010] However, antibody production is a challenging and very expensive process. This is reflected by the high price of antibody products and the even higher costs of antibody therapeutics, limiting their impact on global health issues. These valuable proteins require genetic engineering and manufacturing before they can be used. Researchers or manufacturers are required to isolate antibodies from crude extracts.

[0011] A key step in the isolation of such antibodies is the well-established affinity chromatography using protein A resin. Protein A chromatography is a simple and highly selective method that relies on the strong and specific interaction between protein A and the crystallizable fragment (Fc) of the antibody (Hober S et al., J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Mar 15;848(1):40-7. doi:10.1016 / j.jchromb.2006.09.030.).

[0012] However, the elution step imposes a significant disadvantage for Protein A purification because the target antibody is eluted from the column using a strong shift in buffer pH to acidic conditions. By this means, antibodies can be isolated to high purity, but at the cost of suboptimal buffer conditions and the adverse effects of pH on this type of molecule. These conditions can even exclude sensitive variants or conjugates from purification altogether.

[0013] Alternatives to Protein A, such as cation exchange and non-chromatographic methods like precipitation, have been explored, but they have not been able to compete with the well-established Protein A platform when applied to industrial-scale bioprocessing (Kelley B. MAbs. 2009 Sep-Oct;1(5):443-52. doi:10.4161 / mabs.1.5.9448.). Summary of the Invention [Problem to be solved by the invention]

[0014] Therefore, there is a need in the art for improved affinity matrices that are suitable for fast and easy isolation and / or purification of target molecules. Furthermore, there is a need in the art for improved affinity matrices that reduce contamination and biochemical modification or aggregation of eluted target molecules. Furthermore, there is a need in the art for improved affinity matrices that meet the demands of modern chromatographic processes in terms of short residence times while providing the same binding capacity and reusability. [Means for solving the problem]

[0015] These and other technical problems are solved by the provision of the embodiments as defined herein and as characterized in the claims. According to a first aspect of the present invention, there is provided a photoswitchable azobiaryl compound of formula (I)

[0016] [ka]

[0017] [In the formula, R 1 and R 1’ are independently H, F, Cl, Br, (CH2) n CH3, CH((CH2) n CH3)((CH2) x CH3), C((CH2) n CH3)((CH2)x CH3)((CH2) z CH3), (CH2) n CH((CH2) x CH3)((CH2) z CH3), (CH2) n C((CH2) x CH3)((CH2) y CH3)((CH2) z CH3), O(CH2) n CH3, OCH((CH2) n CH3)((CH2) x CH3), OC((CH2) n CH3)-((CH2) x CH3)((CH2) z CH3), O(CH2) n CH((CH2) x CH3)((CH2) z CH3), O(CH2) n C((CH2) x CH3)-((CH2) y CH3)((CH2) z CH3), S(CH2) n CH3, SCH((CH2) n CH3)((CH2) x CH3), SC((CH2) n CH3)((CH2) x CH3)-((CH2) z CH3), S(CH2) n CH((CH2) x CH3)((CH2) z CH3), S(CH2) n C((CH2) x CH3)((CH2) y CH3)((CH2) z CH3), NH2, NH(CH2) n CH3, NHCH((CH2) n CH3)-((CH2) x CH3), NHC((CH2) n CH3)((CH2) x CH3)((CH2) z CH3), NH(CH2) n CH((CH2) xCH3)((CH2) z CH3), NH(CH2) n C-((CH2) x CH3)-((CH2) y CH3)((CH2) z CH3), N((CH2) n CH3)2, N(CH((CH2) n CH3)((CH2) x CH3))2, N(C((CH2) n CH3)-((CH2) x CH3)((CH2) z CH3))2, N((CH2) n CH((CH2) x CH3)((CH2) z CH3))2, N((CH2) n C((CH2) x CH3)((CH2) y CH3)-((CH2) z CH3))2, aziridin-1-yl, azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, azepan-1-yl, azocan-1-yl, morpholin-1-yl, 4-methyl-piperazin-1-yl, wherein n can be any integer from 0 to 10, x can be any integer from 0 to 10, y can be any integer from 0 to 10, and z can be any integer from 0 to 10; R 2 and R 2’ are independently SO3H, SO3Li, SO3Na, SO3K, COOH, COONHS, CONH2, CONH-(CH2) n CH3, CONH-((CH2) n CH3)((CH2) z CH3), CONH-(CH2) n SO3H, CONH-(CH2) n SO3Li, CONH-(CH2) n SO3Na, CONH-(CH2) n SO3K, CONH-(CH2) n CCH, CONH-(CH2) n N3, CONH-(CH2) nNH2, CONH-(CH2) n COOH, CONH-(CH2) n NHAcI, CONH-(CH2) n NHAcBr, CONH-(CH2) n NHAcCl, CONH-(CH2) n N(maleimide), CONH-(CH2) n -NH(2-chloromethyl acrylate), CONH-(CH2) n -NH(vinyl sulfonate), CONH-(CH2) n -NHCOPhF5, CONH-(CH2) n -NHSO2PhF5, CONH-(CH2) n COONHS, CONH-PEG-OH, CONH-PEG-NH2, CONH-PEG-COOH, CONH-PEG-COONHS, CONH-PEG-O(CH2) n SO3H, CONH-PEG-O(CH2) n SO3Li, CONH-PEG-O(CH2) n SO3Na, CONH-PEG-O(CH2) n SO3K, CONH-PEG-NHAcI, CONH-PEG-NHAcBr, CONH-PEG-NHAcCl, CONH-PEG-N(maleimide), CONH-PEG-NH(2-chloromethyl acrylate), CONH-PEG-NH(vinyl sulfonate), CONH-PEG-NHCOPhF5, CONH-PEG-NHSO2PhF5, CONH-PEG-N3, CONH-PEG-OCH2CCH, CONH-PEG-NHCH2CCH, CONH-PEG-N(CH2CCH)2, CONH-PEG-NHCO(CH2) n COOH, CONH-PEG-NHCO(CH2) n COONHS, CONH-(Xaa) n -OH, CONH-(Xaa) n -NH(CH2) n SO3H, CONH-(Xaa) n -NH(CH2) n SO3Li, CONH-(Xaa) n -NH(CH2) n SO3Na, CONH-(Xaa)n -NH(CH2) n SO3K、CONH-(Xaa) n -OMe、CONH-(Xaa) n -ONHS、CONH-(Xaa) n -ONHS、CONH-(Xaa) n -NH(CH2) n CCH、CONH-(Xaa) n -NH(CH2) n N3、CONH-(Xaa) n -NH(CH2) z NHAcI、CONH-(Xaa) n -NH(CH2) z NHAcBr、CONH-(Xaa) n -NH(CH2) z NHAcCl, CONH-(Xaa) n -NH(CH2) z -N(マレイミド)、CONH-(Xaa) n -NH(CH2) z NH(2-クロロメチルアクリレート)、CONH-(Xaa) n -NH(CH2) z NH(ビニルスルホネート)、CONH-(Xaa) n -NH(CH2) z NHCOPhF5、CONH-(Xaa) n -NH(CH2) z NHSO2PhF5、CONH-(Xaa) n -N3、CONH-(Xaa) n -OCH2CCH、CONH-(Xaa) n -NHCH2CCH、CONH-(Xaa) n -N(OCH2CCH)2、CONH-(Xaa) n -NH-(CH2) n COOH, CONH-(Xaa) n -NH-(CH2) n COONHS、CONH-(Xaa) n -NH-PEG-OH、CONH-(Xaa) n -NH-PEG-NH2、CONH-(Xaa) n -NH-PEG-COOH、CONH-(Xaa)n -NH-PEG-COONHS, CONH-(Xaa) n -NH-PEG-NHAcI, CONH-(Xaa) n -NH-PEG-NHAcBr, CONH-(Xaa) n -NH-PEG-NHAcCl, CONH-(Xaa) n -NH-PEG-N(maleimide), CONH-(Xaa) n -NH-PEG-NH(2-chloromethyl acrylate), CONH-(Xaa) n -NH-PEG-NH(vinyl sulfonate), CONH-(Xaa) n -NH-PEG-NHCOPhF5, CONH-(Xaa) n -NH-PEG-NHSO2PhF5,CONH-(Xaa) n -NH-PEG-N3, CONH-(Xaa) n -NH-PEG-OCH2CCH, CONH-(Xaa) n -NH-PEG-NHCHCCH, CONH-(Xaa) n -NH-PEG-N(CH2CCH)2, CONH-(Xaa) n -NH-PEG-NHCO(CH2) n COOH, CONH-(Xaa) n -NH-PEG-NHCO(CH2) n COONHS, wherein Xaa can be any standard or non-standard amino acid, n can be any integer from 0 to 10, and z can be any integer from 0 to 10; R 3 and R 3’ are independently H, NH2, NHCO(C1-C6-alkyl), NHCO(C1-C6-haloalkyl), NHBoc, NHCbz, NHalloc, NH(CH2) n CH3,NHCH((CH2) n CH3)((CH2) x CH3), NHC((CH2) n CH3)((CH2) x CH3)((CH2) z CH3), NH(CH2) nCH((CH2) x CH3)((CH2) z CH3), NH(CH2) n C((CH2) x CH3)-((CH2) y CH3)((CH2) z CH3), N((CH2) n CH3)2, N(CH((CH2) n CH3)-((CH2) x CH3))2, N(C((CH2) n CH3)((CH2) x CH3)((CH2) z CH3))2, N((CH2) n CH((CH2) x CH3)-((CH2) z CH3))2, N((CH2) n C((CH2) x CH3)((CH2) y CH3)((CH2) z CH3))2, aziridin-1-yl, azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, azepan-1-yl, azocan-1-yl, morpholin-1-yl, 4-methyl-piperazin-1-yl, NHCH2CHCH2, N(CH2CHCH2)2, NHCH2CCH, N(CH2CCH)2, NHCOCCH, NHCO(CH2) n N3, NH acrylate, NH(2-chloromethyl acrylate), NH(vinyl sulfonate), N(maleimide), N(2-bromomaleimide), N(2,3-dibromomaleimide), NHCO aryl, NHCO haloaryl, NHSO2 aryl, NHSO2 haloaryl, (CH2) n N(maleimide), (CH2) n N(2-bromomaleimide), (CH2) n N(2,3-dibromomaleimide), NHCO(CH2) n N(maleimide), NHCO(CH2) n N(2-bromomaleimide), NHCO(CH2) n N(2,3-dibromomaleimide), NCS, NCO, NH(CH2) n CH(O)CH2, N((CH2)n NAcCHCH(O)CH, wherein n can be any integer from 0 to 10, and z can be any integer from 0 to 10. is provided.

[0018] Those skilled in the art will recognize that when a moiety ends in "NHS," the term denotes "N-succinimidyl," derived from N-hydroxysuccinimide, so, for example, COONHS refers to a carboxy N-succinimidyl ester and (Xaa)n-ONHS refers to the C-terminal N-succinimidyl ester of a peptide or amino acid.

[0019] Additionally, "CCH" denotes an alkyne moiety, i.e., a triple bond, so for example, CONH-(CH2) n CCH is CONH-(CH2) n It should be understood that this refers to ethynyl.

[0020] In a preferred embodiment, R 1 or R 1’ At least one of them is not H. In a preferred embodiment of the first aspect of the present invention, aryl 1 -aryl 2 and aryl 1’ -aryl 2’ is a polyfunctionalized biaryl moiety, more preferably wherein at least one R 1 and at least one R 1’ is not hydrogen, and even more preferably both R 1 and both R 1’ But it is not hydrogen.

[0021] It is to be understood that the term "multiply functionalized biaryl moiety" indicates that each moiety bears more than one substituent. 1 -aryl 2 When is a polyfunctionalized biaryl moiety, the two linked aryls contain at least two substituents.

[0022] In another preferred embodiment of the first aspect of the present invention, R 2 and R 2’ at least one, and preferably both, of is CONH-Xaa-OH (Xaa = any standard or non-standard amino acid) or R 2 and R 2’ At least one, and preferably both, of R comprises a linker selected from the group consisting of a peptide, a bifunctional alkane, and a poly(alkylene oxide), more preferably wherein said poly(alkylene oxide) has a molecular weight selected from the group consisting of about 100 g / mol to about 80,000 g / mol, and even more preferably about 100 g / mol to 6,000 g / mol. In another preferred embodiment of the first aspect of the present invention, R 2 or R 2’ At least one of R is not COOH, and preferably both R 2 and R 2’ But it is not COOH.

[0023] The term "about" in the context of the present invention denotes an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question, and typically indicates a deviation of ±5%, preferably ±2%, and more preferably ±1% from the indicated numerical value.

[0024] In one preferred embodiment of the first aspect of the present invention, R 3 and R 3’ at least one, preferably both, of R are substituents selected from the group comprising amines, acrylamides, NHCO(C1-C6-haloalkyl), such as α-haloacetamides, vinylsulfonates, isothiocyanates, isocyanates, epoxides, maleimides, haloarylcarboxy- and haloarylsulfonamides, such as fluorophenylcarboxy- and fluorophenylsulfonamides, more preferably R 3 and R 3’is capable of forming an intramolecular bond within the affinity ligand via an amino acid side chain, and even more preferably, the amino acid side chain comprises a cysteine ​​residue, a histidine residue, a lysine residue, a methionine residue, or any standard or non-standard amino acid in a polypeptide that is accessible for reaction with the ligand-reactive portion of the photoswitchable azobiaryl compound; alternatively, and more preferably, R 3 and R 3’ can form an intermolecular bond between the affinity ligand and a second affinity ligand and / or a solid support via an amino acid side chain, preferably with a cysteine ​​residue, a histidine residue, a lysine residue, a methionine residue, or any standard or non-standard amino acid in a polypeptide or functional group of a solid support that is accessible for reaction with the ligand-reactive moiety of a photoswitchable azobiaryl compound.

[0025] In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-iodoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0026] In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-bromoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-bromoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0027] In another preferred embodiment of the first aspect of the present invention, the compound is 4'-(2-(4-(2-chloroacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0028] In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamide)acetic acid)-4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamide)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamide)acetic acid)-4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamide)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(perfluorobenzamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(perfluorobenzamido))-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-iodoacetamido)-3',5'-diethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-diethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-bromoacetamido)-3',5'-diethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-bromoacetamido)-3',5'-diethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-chloroacetamido)-3',5'-diethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-diethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene.In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-diethoxybiphenyl-3-ylcarboxamide)acetic acid)-4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-diethoxybiphenyl-3-ylcarboxamide)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-diethoxybiphenyl-3-ylcarboxamide)acetic acid)-4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-diethoxybiphenyl-3-ylcarboxamide)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(perfluorobenzamido)-3',5'-diethoxybiphenyl-3-ylcarboxamide)acetic acid)-4'-(2-(4-(perfluorobenzamido))-3',5'-diethoxybiphenyl-3-ylcarboxamide)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-iodoacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-bromoacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-bromoacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene. In another preferred embodiment of the first aspect of the present invention, the compound is 4'-(2-(4-(2-chloroacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene.In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-difluorobiphenyl-3-ylcarboxamide)acetic acid)-4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-difluorobiphenyl-3-ylcarboxamide)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-difluorobiphenyl-3-ylcarboxamide)acetic acid)-4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-difluorobiphenyl-3-ylcarboxamide)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(perfluorobenzamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(perfluorobenzamido))-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-iodoacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-bromoacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-bromoacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-chloroacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)diazene.In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamide)acetic acid)-4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamide)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-dichlorobiphenyl-3-ylcarboxamide)acetic acid)-4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-dichlorobiphenyl-3-ylcarboxamide)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(perfluorobenzamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(perfluorobenzamido))-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-iodoacetamido)-3',5'-dibromobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-dibromobiphenyl-3-ylcarboxamido)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-bromoacetamido)-3',5'-dibromobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-bromoacetamido)-3',5'-dibromobiphenyl-3-ylcarboxamido)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-chloroacetamido)-3',5'-dibromobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-dibromobiphenyl-3-ylcarboxamido)acetic acid)diazene.In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dibromobiphenyl-3-ylcarboxamide)acetic acid)-4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dibromobiphenyl-3-ylcarboxamide)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-dibromobiphenyl-3-ylcarboxamide)acetic acid)-4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-dibromobiphenyl-3-ylcarboxamide)acetic acid)diazene. In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(perfluorobenzamido)-3',5'-dibromobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(perfluorobenzamido))-3',5'-dibromobiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0029] In a preferred embodiment of the first aspect of the present invention, the compound is more compact in the cis state and more extended in the trans state, more preferably wherein adjacent C 4 The distance between cis and trans atoms differs by about 0.5 nm to about 50 nm, even more preferably by about 1 nm to about 30 nm, and most preferably by about 1 nm to about 20 nm.

[0030] In one preferred embodiment of the first aspect of the present invention, the configuration of the photoswitchable azobiaryl compound can be reversibly altered by irradiation with light of a particular wavelength, more preferably the configuration is switched from a cis to a trans state, or alternatively, more preferably the configuration is switched from a trans to a cis state.

[0031] In one preferred embodiment, which is a preceding embodiment of the first aspect of the present invention, the wavelength of the light is at least 400 nm, more preferably at most 750 nm, and even more preferably at most 700 nm.

[0032] In a preferred embodiment of the first aspect of the invention, at least 80% of the compounds are in the trans state when exposed to light of a wavelength between about 400 nm and 490 nm, and at least 80% are in the cis state when exposed to light of a wavelength between about 600 nm and 700 nm.

[0033] In another preferred embodiment of the first aspect of the invention, the compound is soluble in water at pH 8.0 from about 0.001 mM to about 2 mM, more preferably greater than 0.1 mM, and most preferably greater than 1 mM.

[0034] In preferred embodiments of the first aspect of the invention, the thermal half-life of the cis form in water at pH 8.0 at room temperature is from about 1 minute to about 72 hours, more preferably greater than 1 hour, and most preferably greater than 12 hours.

[0035] According to a second aspect of the present invention there is provided a photoswitchable affinity ligand comprising an affinity ligand in stable association with a photoswitchable azobiaryl compound according to the first aspect of the present invention.

[0036] In a preferred embodiment of the second aspect of the invention, the affinity ligand is selected from the group consisting of a peptide, oligopeptide, polypeptide, protein, antibody or antigen-binding fragment thereof, immunoglobulin or fragment thereof, enzyme, hormone, cytokine, complex, oligonucleotide, polynucleotide, nucleic acid, carbohydrate, liposome, nanoparticle, cell, biopolymer, biomolecule or small molecule.

[0037] In one preferred embodiment of the second aspect of the invention, the photoswitchable azobiaryl compound is stably associated in a bifunctional manner with two conjugation sites within the affinity ligand.

[0038] In another preferred embodiment of the second aspect of the invention, exposure to light of a specific wavelength induces a conformational switch, causing a loss of the specific affinity of the photoswitchable affinity ligand for the target molecule.

[0039] In a preferred embodiment of the second aspect of the invention, the affinity ligand is selected from the group comprising immunoglobulin (Ig) binding proteins, more preferably from the group comprising protein A, protein G and protein L, or variants thereof capable of specifically binding to immunoglobulins.

[0040] In a preferred embodiment of the second aspect of the invention, the affinity ligand has none, only one or a defined set of lysine residues for site-specific immobilization to a solid phase.

[0041] In one preferred embodiment of the second aspect of the present invention, the affinity ligand comprises the B domain of protein A (SEQ ID NO: 4), which may have two substitutions of wild-type residues by cysteines, or comprises a protein domain having at least 80% sequence identity with SEQ ID NO: 4; more preferably, the affinity ligand comprises a protein domain having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7; even more preferably, the affinity ligand comprises a protein domain having the amino acid sequence of SEQ ID NO: 7.

[0042] According to a preferred embodiment of the present invention, the dissociation constant (KD) for binding of an affinity ligand as envisaged herein to a target molecule is in the sub-nM to mM range, preferably at most 1000 nM, more preferably at most 100 nM.

[0043] In another preferred embodiment of the second aspect of the present invention, the affinity ligand comprises the lysine-poor B domain of protein A (SEQ ID NO: 5), optionally substituted with two cysteine ​​residues, or comprises a protein domain having at least 80% sequence identity with SEQ ID NO: 5; more preferably, the affinity ligand comprises a protein domain having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11; even more preferably, the affinity ligand comprises a protein domain having the amino acid sequence of SEQ ID NO: 11.

[0044] In one preferred embodiment of the second aspect of the invention, the affinity ligand comprises at least one of the three homologous domains of protein G defined as C1 (SEQ ID NO: 12), C2 (SEQ ID NO: 13) and C3 (SEQ ID NO: 14), optionally substituted with two cysteine ​​residues, or a protein domain having at least 80% sequence identity with SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14. Preferably, the affinity ligand comprises C1, optionally substituted with two cysteine ​​residues.

[0045] In another preferred embodiment of the second aspect of the invention, the affinity ligand comprises at least one lysine-deficient C1 domain of protein G (SEQ ID NO: 15), optionally substituted with two cysteine ​​residues, or a protein domain having at least 80% sequence identity with SEQ ID NO: 15.

[0046] In yet another preferred embodiment of the second aspect of the invention, the affinity ligand comprises at least one variant of the lysine-poor C1 domain of protein G (SEQ ID NO: 18) or a protein domain having at least 80% sequence identity with SEQ ID NO: 18.

[0047] Within the present disclosure, lysine deficiency is intended to indicate that the lysine present in a given amino acid sequence has been replaced with any other amino acid. In one preferred embodiment of the second aspect of the present invention, the affinity ligand comprises the C domain of protein L, which may be substituted with two cysteine ​​residues, or a protein domain having at least 80% sequence identity thereto; more preferably, the C domain of protein L is the C2 domain of protein L (SEQ ID NO: 16) or a protein domain having at least 80% sequence identity thereto.

[0048] In another preferred embodiment of the second aspect of the invention, the affinity ligand comprises the lysine-deficient C2 domain of protein L (SEQ ID NO: 17), optionally substituted with two cysteine ​​residues, or a protein domain having at least 80% sequence identity thereto. In yet another preferred embodiment of the second aspect of the invention, the affinity ligand comprises a variant of the lysine-deficient C2 domain of protein L (SEQ ID NO: 19), or a protein domain having at least 80% sequence identity thereto.

[0049] According to a third aspect of the present invention, there is provided a photoswitchable affinity matrix comprising a solid support and a photoswitchable azobiaryl compound according to the first aspect of the invention in stable association with an affinity ligand, wherein the photoswitchable azobiaryl compound and affinity ligand are in stable association with the solid support, or a photoswitchable affinity ligand according to the second aspect of the invention in stable association with the solid support.

[0050] In another preferred embodiment of the third aspect of the invention, the photoswitchable affinity ligand is stably associated with the solid support via site-specific binding. According to a fourth aspect of the present invention there is provided the use of a photoswitchable compound for isolating and / or purifying a target molecule.

[0051] In preferred embodiments of the invention, particularly of the fourth aspect of the invention, the compound is not 4'-carboxyphenylazophenylalanine, more preferably the compound is not 3'-carboxyphenylazophenylalanine or 4'-carboxyphenylazophenylalanine, and even more preferably the compound is not 3'-carboxyphenylazophenylalanine or a derivative thereof, or 4'-carboxyphenylazophenylalanine or a derivative thereof.

[0052] In a preferred embodiment of the fourth aspect of the invention, the photoswitchable compound is a photoswitchable azobiaryl compound according to the first aspect of the invention. According to a fifth aspect of the present invention there is provided the use of a photoswitchable affinity ligand, comprising an affinity ligand in stable association with a photoswitchable compound, for isolating and / or purifying a target molecule.

[0053] In a preferred embodiment of the fifth aspect of the present invention, the photoswitchable compound is not 4'-carboxyphenylazophenylalanine, more preferably the compound is not 3'-carboxyphenylazophenylalanine or 4'-carboxyphenylazophenylalanine, even more preferably the compound is not 3'-carboxyphenylazophenylalanine or a derivative thereof or 4'-carboxyphenylazophenylalanine or a derivative thereof.

[0054] In a preferred embodiment of the fifth aspect of the invention, the photoswitchable affinity ligand is a photoswitchable affinity ligand according to the first aspect of the invention. According to a sixth aspect of the present invention there is provided the use of a photoswitchable affinity matrix according to the third aspect of the present invention for isolating and / or purifying a target molecule.

[0055] In a preferred embodiment of the fourth, fifth or sixth aspect of the invention, the target molecule is an immunoglobulin, more preferably the target molecule is an Fc domain containing immunoglobulin, even more preferably an IgG type immunoglobulin.

[0056] According to a seventh aspect of the present invention, there is provided a method for isolating and / or purifying a target molecule, the method comprising the steps of: providing a composition comprising the target molecule; contacting the composition with an affinity matrix for a time sufficient to allow specific binding of the target molecule to the affinity matrix, the affinity matrix comprising a photoswitchable compound stably associated with an affinity ligand and a solid support to form an affinity matrix; washing the affinity matrix with a wash solution to remove components of the composition that are not specifically bound to the affinity matrix; irradiating the affinity matrix with light of a wavelength of at least about 400 nm to cause loss of specific binding of the affinity matrix to the target molecule; and eluting the target molecule from the affinity matrix using an eluent.

[0057] According to an eighth aspect of the present invention, there is provided a method for isolating and / or purifying a target molecule, the method comprising the steps of: providing a composition comprising the target molecule; contacting the composition with an affinity matrix for a time sufficient to allow specific binding of the target molecule to the affinity matrix, the affinity matrix comprising a photoswitchable azobiaryl compound comprising at least two substituted biphenyl moieties, preferably a photoswitchable azobiaryl compound according to the first aspect of the present invention in stable association with an affinity ligand, wherein the photoswitchable azobiaryl compound and the affinity ligand comprise a photoswitchable azobiaryl compound in stable association with a solid support to form an affinity matrix, or a photoswitchable affinity ligand according to the second aspect of the present invention in stable association with a solid support to form an affinity matrix, or a photoswitchable affinity matrix according to the third aspect of the present invention; washing the affinity matrix with a wash solution to remove components of the composition that are not specifically bound to the affinity matrix; altering irradiation of the affinity matrix with light of a particular wavelength to cause the affinity matrix to lose its specific binding or affinity for the target molecule; and eluting the target molecule from the affinity matrix using an eluent. [Brief explanation of the drawings]

[0058] [Figure 1]1 shows photoswitchable azobiaryl compounds according to the present invention. (A) Basic chemical structure of a photoswitchable azobiaryl compound, including an azobenzene core and peripheral groups R, R, R, and R′, R, R′ as defined in accordance with the present invention; (B) Chemical structure of a preferred photoswitchable azobiaryl compound, 4′-(2-(4-(2-iodoacetamido)-3′,5′-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4′-(2-(4-(2-iodoacetamido)-3′,5′-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene, herein referred to as PS1; (C) Atomic distances at the p-positions of PS1 in the extended trans state (24.8A) and (D) the more compact cis state (12.9A). [Figure 2] FIG. 2 shows the synthesis of (A) the photoswitchable azobiaryl compound PS1 and (B) the MnIII-SalophenCl catalyzer. [Figure 3] Figure 3 shows (A) the coupling of the photoswitchable azobiaryl compound PS1 with N-acetylcysteine ​​and photoswitching of the reaction product with alternating irradiation cycles, (B) HPLC analysis of PS1 coupled with N-acetylcysteine ​​after photoswitching (isomerization) with 465 nm (blue) and 635 nm (red) irradiation, and (C) the UV-VIS spectrum of the photoswitchable azobiaryl compound PS1 coupled with N-acetylcysteine ​​in aqueous buffer (solid line: trans isomer; dotted line: cis isomer). [Figure 4] FIG. 4 shows the ESI-MS spectra of unmodified SpA#1 (upper panel) and PS1-modified SpA#1 (lower panel). [Figure 5]Figure 5 shows (A) photo-controlled affinity chromatography of an IgG sample using SpA#1-PS1; (B) SDS-PAGE analysis of the raw material and elution fractions. Both samples showed characteristic bands for the heavy (approximately 50 kDa) and light (approximately 25 kDa) chains of the IgG molecule, indicating specific binding and subsequent photo-controlled elution of immunoglobulin G. The isolated IgG was also analyzed by analytical SEC (C) and nano-differential scanning fluorimetry (nano-DSF) (D) using a Tycho NT.6 (NanoTemper Technologies). After purification using the SpA#1-PS1 affinity matrix, the results indicated high-quality protein. (E) Proposed concept of photo-controlled affinity modulation. The effect of affinity modification is most likely the result of distortion of the protein-ligand secondary structure upon photoisomerization of the photoswitch PS1. [Figure 6] Figure 6 shows chromatographic profiles illustrating affinity purification of an immunoglobulin G (IgG) mixture (Cutaquig®) using photoswitchable protein variants: (A) chromatogram of the purification process using a protein G variant, SpG#1, modified with the azobiaryl-based photoswitch PS2; (B) chromatogram of the purification process using a protein L variant, PpL#1, also modified with PS2. Both panels demonstrate sample application and subsequent washing steps under red light illumination. Elution of specifically bound IgG molecules is initiated by blue light illumination, as indicated by arrows in each chromatogram. This figure effectively illustrates the controlled manipulation of binding and release events in the purification process via light-responsive protein engineering. [Figure 7]Figure 7 shows (A) an HPLC analysis demonstrating the photoswitching behavior of the azobiaryl compound PS4 over successive irradiation cycles, including periods in the dark after exposure to 593 nm (yellow light), 450 nm (blue light), and 312 nm (UV light) irradiation. This analysis illustrates the reversible transition of PS4 under different wavelengths of light, highlighting its photoresponsive properties. (B) UV-VIS spectrum of the photoswitchable azobiaryl compound PS4, where the solid line represents the trans isomer and the dotted line represents the cis isomer. These spectra provide insight into the optical properties of PS4, detailing the absorption differences between its isomers and supporting its utility in photoswitching applications. DETAILED DESCRIPTION OF THE INVENTION

[0059] Before the present invention is described in more detail below, it is to be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0060] The present invention is based on the identification of the basic chemical structure of photoswitchable azobiaryl compounds that can stably associate with affinity ligands to enable digital switching of the affinity ligand between high and low affinity with a protein of interest by using light of different wavelengths. Particular advantages of the compounds of the present invention include their additional solubility in aqueous solutions compared to similar molecules of the prior art that are soluble only in organic solvents, thus preventing their functionalization without interfering with the function of the affinity ligand.

[0061] These advantages are provided by the structure of the photoswitchable azobenzene compounds according to the present invention, wherein R1 and R 1 ' facilitates a bathochromic shift in wavelength from trans to cis isomerization, and R 2 and R 2 ' is a solubility-enhancing moiety and R 3 and R 3 ' is a ligand-reactive and / or solid support-reactive moiety.

[0062] Kohl et al. (Synthesis 2014, 46, 2376-2382) disclose azobiaryl compounds that are structurally similar to the compounds of the present invention, however, the compounds disclosed therein are used in different and unrelated technical fields and also have different substitution patterns than the compounds of the present invention, thus resulting in a structure that is distinct from the compounds of the present invention as described herein.

[0063] As in traditional methods of affinity purification, target molecules are effectively captured from heterogeneous protein compositions by immobilized photoswitchable affinity ligands of the present invention. However, using the compounds of the present invention, the affinity of affinity ligands associated with photoswitchable azobiaryl compounds for a target can be digitally switched using different wavelengths of light.

[0064] By this means, molecules can be isolated in a selected buffer to mitigate their sensitive nature and eliminate the need for a buffer exchange procedure in the next process step. Furthermore, the mode of operation allows for rapid chromatography cycles with dramatically reduced buffer consumption, which implies the potential for scalability and significant savings. Due to the elimination of the buffer-induced elution step and the fast regeneration of the affinity matrix after elution, the effective binding capacity and productivity of the photoswitchable affinity chromatography system are clearly superior to processes known in the art.

[0065] Host cell protein (HCP) contaminant clearance is a significant concern during affinity chromatography (Wolter, T. and Richter, A. (2005). Assays for controlling host-cell impurities in biopharmaceuticals. BioProcess Int, 3(2), 40-46). Conventionally, an intermediate pH wash is used between column loading and elution to minimize HCP levels during elution. HCP contaminants that co-elute at low pH include species that associate with the product and / or the chromatographic resin. By eliminating the step of changing buffer conditions, only specifically bound target molecules are eluted using a photoswitchable chromatography matrix.

[0066] Photoswitches are molecules that can be reversibly interconverted between (at least) two states with the aid of light irradiation. Azobenzenes remain one of the most ubiquitous photoswitches due to their stability, reliability, and tunability; they offer high extinction coefficients and quantum yields, allowing switching between cis and trans isomers with low light intensities, and are stable to repeated switching cycles.

[0067] With regard to optical control of affinity matrices, there are several performance indicators that can be used to qualify the azobenzene core as a molecular optical switch. - Cis to trans and trans to cis isomerization wavelengths - Thermal stability of cis isomer - switching integrity at a given wavelength - Change in end-to-end distance upon photoisomerization (shape change) Most azobenzene-based photoswitches developed to date require the use of UV light for photoisomerization from the trans to the cis state. This limits their use to non-research and industrial-scale applications such as affinity chromatography, primarily because UV light is strongly scattered, making it difficult to penetrate materials used as solid supports, such as agarose beads or polymer-based membranes. Furthermore, more energetic UV light can induce degradation and chemical modification of these materials.

[0068] Also, known and previously explored photoswitches in the art appear to be soluble only in polar to nonpolar organic solvents and insoluble in aqueous solutions. Clearly, this renders known photoswitches unusable in the aqueous environments commonly used in the purification of therapeutics, such as therapeutic antibodies, where the crude extract, affinity ligand, and protein of interest are water soluble.

[0069] Therefore, a shift in the wavelength of isomerization toward the visible light region (bathochromic shift) is desirable. From the energy perspective, the trans isomer of an azobenzene compound has a lower intrinsic energy than the cis isomer. Therefore, the azo structure spontaneously recovers from the cis state to the trans state. Therefore, the cis state has poorer thermal stability and a shorter lifetime due to its higher energy state.

[0070] The short-lived cis isomer means that an intense light source is required to maintain a significant proportion of the cis isomer, which is an undesirable limitation to its application in affinity chromatography. To achieve high cis-to-trans isomerization efficiency, the two isomers of the azobenzene derivative must provide well-separated absorption bands. Most frequently, trans-to-cis photoisomerization occurs via a high-energy π-π bond in the trans isomer. * cis to trans photoisomerization is achieved by irradiating the region of the n-π band of the cis isomer, whereas cis to trans photoisomerization is achieved by irradiating the region of the n-π band of the cis isomer, *This occurs through irradiation in the band (Merino, E. and Ribagorda, M. (2012). Control over molecular motion using the cis-trans photoisomerization of the azo group. Beilstein Journal of Organic Chemistry, 8(1), 1071-1090.).

[0071] However, the overlap in absorbance between cis and trans isomers leads to incomplete photoswitching. To exploit and / or amplify the end-to-end distance to larger length scales upon photoisomerization, it is essential to embed the azobenzene core in a conformationally rigid molecular scaffold. To this end, a viable strategy consists of incorporating them into rigid aromatic structures, such as biaryls.

[0072] The wavelength of cis to trans isomerization, thermal stability, phototransformation, as well as shape variation, strongly depend on the substitution pattern of the azobenzene core. Azobenzene photoswitches have been used in organic synthesis (Wolf, E. and Cammenga, HK (1977). Thermodynamic and kinetic investigation of the thermal isomerization of cis-azobenzene. Zeitschrift fur Physikalische Chemie, 107(1), 21-38.), functional materials including self-healing materials (Suginome, H. (2004). CRC Handbook of Organic Photochemistry and Photobiology.), adhesives (Morgenstern, K. (2009). Isomerization reactions on single adsorbed molecules. Accounts of chemical research, 42(2), 213-223.), and photoresists (Pace, G., Ferri, V., Grave, C., Elbing, M., von Hanisch, C., Zharnikov, M., ... and Samori, P. (2007). Cooperative light-induced molecular movements of highly ordered azobenzene self-assembled They have previously been reported to be used in polymers as monolayers. Proceedings of the National Academy of Sciences, 104(24), 9937-9942.), and optical materials (Choi, BY, Kahng, SJ, Kim, S., Kim, H., Kim, HW, Song, YJ, ... and Kuk, Y. (2006). Conformational molecular switch of the azobenzene molecule: a scanning tunneling microscopy study. Physical review letters, 96(15), 156-106.). In these applications, they have been used in solid / liquid states or as solutions in organic solvents.

[0073] However, there is a particular need and interest in photoswitches that are functional in aqueous solutions, e.g., for modulation of biological activity. Due to the inherently hydrophobic character of azobenzene derivatives, the modification of molecules with such photoswitches has been limited to reactions that can be carried out in organic solvents or organic solvent / water mixtures.

[0074] However, sensitive biomolecules such as polypeptides or proteins can irreversibly unfold under these conditions. An intact 3D structure of the polypeptide or protein is a requirement to address the correct binding site, and therefore an aqueous buffer system is necessary to allow stable association of the photoswitch.

[0075] This technical problem is solved by the compounds of the present invention that incorporate a solubility-enhancing moiety as described herein. The solubility-enhanced photoswitchable azobiaryl compounds have good solubility in aqueous solutions at physiological pH, which represents one of the technical advantages of the present invention.

[0076] Thus, the photoswitchable azobiaryl compounds defined by formula (I) as provided herein have the advantage that the critical aspects discussed above can be addressed to meet the stringent demands for affinity chromatography by appropriate substituent selection.

[0077] Thus, in a first aspect of the present invention, there is provided a photoswitchable azobiaryl compound of formula (I)

[0078] [ka]

[0079] [In the formula, R 1 and R 1’ are independently H, F, Cl, Br, (CH2) n CH3, CH((CH2) n CH3)((CH2) x CH3), C((CH2) nCH3)((CH2) x CH3)((CH2) z CH3), (CH2) n CH((CH2) x CH3)((CH2) z CH3), (CH2) n C((CH2) x CH3)((CH2) y CH3)((CH2) z CH3), O(CH2) n CH3, OCH((CH2) n CH3)((CH2) x CH3), OC((CH2) n CH3)-((CH2) x CH3)((CH2) z CH3), O(CH2) n CH((CH2) x CH3)((CH2) z CH3), O(CH2) n C((CH2) x CH3)-((CH2) y CH3)((CH2) z CH3), S(CH2) n CH3, SCH((CH2) n CH3)((CH2) x CH3), SC((CH2) n CH3)((CH2) x CH3)-((CH2) z CH3), S(CH2) n CH((CH2) x CH3)((CH2) z CH3), S(CH2) n C((CH2) x CH3)((CH2) y CH3)((CH2) z CH3), NH2, NH(CH2) n CH3, NHCH((CH2) n CH3)-((CH2) x CH3), NHC((CH2) n CH3)((CH2) x CH3)((CH2) z CH3), NH(CH2) n CH((CH2)x CH3)((CH2) z CH3), NH(CH2) n C-((CH2) x CH3)-((CH2) y CH3)((CH2) z CH3), N((CH2) n CH3)2, N(CH((CH2) n CH3)((CH2) x CH3))2, N(C((CH2) n CH3)-((CH2) x CH3)((CH2) z CH3))2, N((CH2) n CH((CH2) x CH3)((CH2) z CH3))2, N((CH2) n C((CH2) x CH3)((CH2) y CH3)-((CH2) z CH3))2, aziridin-1-yl, azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, azepan-1-yl, azocan-1-yl, morpholin-1-yl, 4-methyl-piperazin-1-yl, wherein n can be any integer from 0 to 10, x can be any integer from 0 to 10, y can be any integer from 0 to 10, and z can be any integer from 0 to 10; R 2 and R 2’ are independently SO3H, SO3Li, SO3Na, SO3K, COOH, COONHS, CONH2, CONH-(CH2) n CH3, CONH-((CH2) n CH3)((CH2) z CH3), CONH-(CH2) n SO3H, CONH-(CH2) n SO3Li, CONH-(CH2) n SO3Na, CONH-(CH2) n SO3K, CONH-(CH2) n CCH, CONH-(CH2) n N3, CONH-(CH2)n NH2, CONH-(CH2) n COOH, CONH-(CH2) n NHAcI, CONH-(CH2) n NHAcBr, CONH-(CH2) n NHAcCl, CONH-(CH2) n N(maleimide), CONH-(CH2) n -NH(2-chloromethyl acrylate), CONH-(CH2) n -NH(vinyl sulfonate), CONH-(CH2) n -NHCOPhF5, CONH-(CH2) n -NHSO2PhF5, CONH-(CH2) n COONHS, CONH-PEG-OH, CONH-PEG-NH2, CONH-PEG-COOH, CONH-PEG-COONHS, CONH-PEG-O(CH2) n SO3H, CONH-PEG-O(CH2) n SO3Li, CONH-PEG-O(CH2) n SO3Na, CONH-PEG-O(CH2) n SO3K, CONH-PEG-NHAcI, CONH-PEG-NHAcBr, CONH-PEG-NHAcCl, CONH-PEG-N(maleimide), CONH-PEG-NH(2-chloromethyl acrylate), CONH-PEG-NH(vinyl sulfonate), CONH-PEG-NHCOPhF5, CONH-PEG-NHSO2PhF5, CONH-PEG-N3, CONH-PEG-OCH2CCH, CONH-PEG-NHCH2CCH, CONH-PEG-N(CH2CCH)2, CONH-PEG-NHCO(CH2) n COOH, CONH-PEG-NHCO(CH2) n COONHS, CONH-(Xaa) n -OH, CONH-(Xaa) n -NH(CH2) n SO3H, CONH-(Xaa) n -NH(CH2) n SO3Li, CONH-(Xaa) n -NH(CH2) nSO3Na,CONH-(Xaa) n -NH(CH2) n SO3K、CONH-(Xaa) n -OMe、CONH-(Xaa) n -ONHS、CONH-(Xaa) n -ONHS、CONH-(Xaa) n -NH(CH2) n CCH、CONH-(Xaa) n -NH(CH2) n N3、CONH-(Xaa) n -NH(CH2) z NHAcI、CONH-(Xaa) n -NH(CH2) z NHAcBr、CONH-(Xaa) n -NH(CH2) z NHAcCl, CONH-(Xaa) n -NH(CH2) z -N(マレイミド)、CONH-(Xaa) n -NH(CH2) z NH(2-クロロメチルアクリレート)、CONH-(Xaa) n -NH(CH2) z NH(ビニルスルホネート)、CONH-(Xaa) n -NH(CH2) z NHCOPhF5、CONH-(Xaa) n -NH(CH2) z NHSO2PhF5、CONH-(Xaa) n -N3、CONH-(Xaa) n -OCH2CCH、CONH-(Xaa) n -NHCH2CCH、CONH-(Xaa) n -N(OCH2CCH)2、CONH-(Xaa) n -NH-(CH2) n COOH, CONH-(Xaa) n -NH-(CH2) n COONHS、CONH-(Xaa) n -NH-PEG-OH、CONH-(Xaa) n -NH-PEG-NH2、CONH-(Xaa) n-NH-PEG-COOH, CONH-(Xaa) n -NH-PEG-COONHS, CONH-(Xaa) n -NH-PEG-NHAcI, CONH-(Xaa) n -NH-PEG-NHAcBr, CONH-(Xaa) n -NH-PEG-NHAcCl, CONH-(Xaa) n -NH-PEG-N(maleimide), CONH-(Xaa) n -NH-PEG-NH(2-chloromethyl acrylate), CONH-(Xaa) n -NH-PEG-NH(vinyl sulfonate), CONH-(Xaa) n -NH-PEG-NHCOPhF5, CONH-(Xaa) n -NH-PEG-NHSO2PhF5,CONH-(Xaa) n -NH-PEG-N3, CONH-(Xaa) n -NH-PEG-OCH2CCH, CONH-(Xaa) n -NH-PEG-NHCHCCH, CONH-(Xaa) n -NH-PEG-N(CH2CCH)2, CONH-(Xaa) n -NH-PEG-NHCO(CH2) n COOH, CONH-(Xaa) n -NH-PEG-NHCO(CH2) n COONHS, wherein Xaa can be any standard or non-standard amino acid, n can be any integer from 0 to 10, and z can be any integer from 0 to 10; R 3 and R 3’ are independently H, NH2, NHCO(C1-C6-alkyl), NHCO(C1-C6-haloalkyl), NHBoc, NHCbz, NHalloc, NH(CH2) n CH3,NHCH((CH2) n CH3)((CH2) x CH3), NHC((CH2) n CH3)((CH2) x CH3)((CH2) zCH3), NH(CH2) n CH((CH2) x CH3)((CH2) z CH3), NH(CH2) n C((CH2) x CH3)((CH2) y CH3)((CH2) z CH3), N((CH2) n CH3)2, N(CH((CH2) n CH3)-((CH2) x CH3))2, N(C((CH2) n CH3)((CH2) x CH3)((CH2) z CH3))2, N((CH2) n CH((CH2) x CH3)-((CH2) z CH3))2, N((CH2) n C((CH2) x CH3)((CH2) y CH3)((CH2) z CH3))2, aziridin-1-yl, azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, azepan-1-yl, azocan-1-yl, morpholin-1-yl, 4-methyl-piperazin-1-yl, NHCH2CHCH2, N(CH2CHCH2)2, NHCH2CCH, N(CH2CCH)2, NHCOCCH, NHCO(CH2) n N3, NH acrylate, NH(2-chloromethyl acrylate), NH(vinyl sulfonate), N(maleimide), N(2-bromomaleimide), N(2,3-dibromomaleimide), NHCO aryl, NHCO haloaryl, NHSO2 aryl, NHSO2 haloaryl, (CH2) n N(maleimide), (CH2) n N(2-bromomaleimide), (CH2) n N(2,3-dibromomaleimide), NHCO(CH2) n N(maleimide), NHCO(CH2) n N(2-bromomaleimide), NHCO(CH2) n N(2,3-dibromomaleimide), NCS, NCO, NH(CH2)n CH(O)CH2, N((CH2) n NAcCHCH(O)CH, wherein n can be any integer from 0 to 10, and z can be any integer from 0 to 10. is provided.

[0080] In a preferred embodiment, R 1 or R 1’ At least one of them is not H. In one embodiment of the first aspect of the present invention, R 1 and R 1’ are independently H, F, Cl, Br, (CH2) m CH3, CH((CH2) n CH3)((CH2) x CH3), C((CH2) n CH3)((CH2) x CH3)((CH2) z CH3), (CH2) n CH((CH2) x CH3)((CH2) z CH3), (CH2) n C((CH2) x CH3)((CH2) y CH3)((CH2) z CH3), O(CH2) n CH3, OCH((CH2) n CH3)((CH2) x CH3), OC((CH2) n CH3)-((CH2) x CH3)((CH2) z CH3), O(CH2) n CH((CH2) x CH3)((CH2) z CH3), O(CH2) n C((CH2) x CH3)-((CH2) y CH3)((CH2) z CH3), S(CH2) n CH3, SCH((CH2) n CH3)((CH2)x CH3), SC((CH2) n CH3)((CH2) x CH3)-((CH2) z CH3), S(CH2) n CH((CH2) x CH3)((CH2) z CH3), S(CH2) n C((CH2) x CH3)((CH2) y CH3)((CH2) z CH3), NH2, NH(CH2) n CH3, NHCH((CH2) n CH3)-((CH2) x CH3), NHC((CH2) n CH3)((CH2) x CH3)((CH2) z CH3), NH(CH2) n CH((CH2) x CH3)((CH2) z CH3), NH(CH2) n C-((CH2) x CH3)-((CH2) y CH3)((CH2) z CH3), N((CH2) n CH3)2, N(CH((CH2) n CH3)((CH2) x CH3))2、N(C((CH2) n CH3)-((CH2) x CH3)((CH2) z CH3))2、N((CH2) n CH((CH2) x CH3)((CH2) z CH3))2、N((CH2) n C((CH2) x CH3)((CH2) y CH3)-((CH2) zCH3))2, aziridin-1-yl, azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, azepan-1-yl, azocan-1-yl, morpholin-1-yl, 4-methyl-piperazin-1-yl, wherein m can be any integer from 1 to 10, n can be any integer from 0 to 10, x can be any integer from 0 to 10, y can be any integer from 0 to 10, and z can be any integer from 0 to 10.

[0081] In one embodiment of the first aspect of the present invention, R 3 and R 3’ are independently H, NH2, NHAc, NHBoc, NHCbz, NHalloc, NHTfAc, NH(CH2) n CH3,NHCH((CH2) n CH3)((CH2) x CH3), NHC((CH2) n CH3)((CH2) x CH3)-((CH2) z CH3), NH(CH2) n CH((CH2) x CH3)((CH2) z CH3), NH(CH2) n C((CH2) x CH3)((CH2) y CH3)((CH2) z CH3), N((CH2) n CH3)2, N(CH((CH2) n CH3)-((CH2) x CH3))2, N(C((CH2) n CH3)((CH2) x CH3)((CH2) z CH3))2, N((CH2) n CH((CH2) x CH3)-((CH2) z CH3))2, N((CH2) n C((CH2) x CH3)((CH2) y CH3)((CH2) zCH3))2, aziridin-1-yl, azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, azepan-1-yl, azocan-1-yl, morpholin-1-yl, 4-methyl-piperazin-1-yl, NHCH2CHCH2, N(CH2CHCH2)2, NHCH2CCH, N(CH2CCH)2, NHCOCCH, NHCO(CH2) n N3, NHAcCl, NHAcBr, NHAcI, NHAcrylate, NH(2-chloromethylacrylate), NH(vinylsulfonate), N(maleimide), N(2-bromomaleimide), N(2,3-dibromomaleimide), NHCOPhF5, NHSO2PhF5, (CH2) n N(maleimide), (CH2) n N(2-bromomaleimide), (CH2) n N(2,3-dibromomaleimide), NHCO(CH2) n N(maleimide), NHCO(CH2) n N(2-bromomaleimide), NHCO(CH2) n N(2,3-dibromomaleimide), NCS, NCO, NH(CH2) n CH(O)CH2, N((CH2) n NAcCHCH(O)CH, wherein n can be any integer from 0 to 10 and z can be any integer from 0 to 10.

[0082] The term "alkyl," as used herein, denotes, in each instance, a straight-chain or branched-chain alkyl group, typically having from 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and more preferably 1 to 3 or 1 or 2 carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, isobutyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, and 1-ethyl-2-methylpropyl.

[0083] The term "haloalkyl," as used herein, denotes in each instance a straight-chain or branched alkyl group, usually having from 1 to 6 carbon atoms, frequently from 1 to 4 carbon atoms, and preferably from 1 to 3 or 1 or 2 carbon atoms, in which the hydrogen atoms of the group are partially or entirely replaced by halogen atoms, such as fluorine, bromine, chlorine, or iodine. Preferred haloalkyl moieties are selected from C-C-haloalkyl, more preferably C-C-haloalkyl or C-C-haloalkyl, and in particular C-C-fluoroalkyl, such as fluoromethyl, bromomethyl, chloromethyl, iodomethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, and the like.

[0084] The term "aryl," as used herein, refers to an "aromatic ring system" (i.e., having -(4n+π2) electrons that satisfy Huckel's rule, where n is 0 or, preferably, an integer from 1 to 3). More specifically, these aromatic ring systems may be monocyclic, bicyclic, or tricyclic, having 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ring carbon atoms. Even more specifically, these aromatic ring systems may be monocyclic, having 6 ring carbon atoms. Exemplary aryl groups are phenyl, biphenyl, naphthyl, anthracyl, and the like.

[0085] The term "haloaryl," as used herein, refers to an aryl in which the hydrogen atoms of the group are partially or totally replaced with halogen atoms such as fluorine, bromine, chlorine, or iodine. Exemplary haloaryls are pentafluorophenyl, monofluorophenyl (ortho, meta, para), and the like.

[0086] According to the present invention, regardless of the representation of a compound in its cis or trans form, it is considered that other isomeric forms are also envisaged unless expressly indicated to the contrary. In a preferred embodiment of the first aspect of the present invention, aryl 1 -aryl 2 and aryl 1’ -aryl 2’ is a polyfunctionalized biaryl compound, more preferably having at least one R 1 and at least one R 1’ is not hydrogen, and even more preferably both R 1 and both R 1’ is not hydrogen. In a preferred embodiment of the first aspect of the present invention, R 1 or R 1’ At least one of the R 1 and at least one R 1’ is not methyl or both R 1 and both R 1’ is not methyl. Preferably, aryl 1 -aryl2 may have up to four substituents, more preferably exactly four substituents. Also preferably, aryl 1’ -aryl 2’ may have at most four substituents, more preferably exactly four substituents. Particularly preferably, each biaryl may have at most four substituents, more preferably exactly four substituents.

[0087] In one preferred embodiment, R 1 and R 1’ is independently selected from the group comprising alkoxy or halogen, more preferably from the group comprising methoxy, ethoxy, chlorine or fluorine.

[0088] In another preferred embodiment of the first aspect of the present invention, R 2 and R 2’ at least one, and preferably both, of is CONH-Xaa-OH (Xaa = any standard or non-standard amino acid) or R 2 and R 2’ At least one, and preferably both, of R comprises a linker selected from the group consisting of a peptide, a bifunctional alkane, and a poly(alkylene oxide), more preferably wherein said poly(alkylene oxide) has a molecular weight selected from the group consisting of about 100 g / mol to about 80,000 g / mol, and even more preferably about 100 g / mol to 6,000 g / mol. In another preferred embodiment of the first aspect of the present invention, R 2 or R 2’ At least one of R is not COOH, and preferably both R 2 and R 2’ But it is not COOH.

[0089] In one preferred embodiment, R 2 and / or R 2’ is an amide or peptide, more preferably -CONH-(Xaa) n It may be -OH, and even more preferably -COHNH-CH2-COOH.

[0090] In one preferred embodiment of the first aspect of the present invention, R 3 and R 3’ At least one, and preferably both, of the formula (I) are substituents selected from the group comprising amines, acrylamides, NHCO(C1-C6-haloalkyl), such as α-haloacetamides, vinylsulfonates, isothiocyanates, isocyanates, epoxides, maleimides, haloarylcarboxy- and haloarylsulfonamides, such as fluorophenylcarboxy- and fluorophenylsulfonamides.

[0091] Preferably, R 3 and / or R 3’ is generally a substituent that is reactive towards one or more of an affinity ligand, a solid support, a polymer, a polypeptide, an oligonucleotide, a polynucleotide, a nucleic acid, a carbohydrate, a liposome, a nanoparticle, a cell, a biopolymer, a biomolecule, or a small molecule.

[0092] R 3 and / or R 3’ The ligand-reactive moiety of is preferably any of a variety of reactive groups that provide a stable association of the photoswitchable azobiaryl compound with the affinity ligand. Stable association of the photoswitchable azobiaryl compound with the affinity ligand includes covalent bonding as well as non-covalent associations such as ionic interactions. Preferably, the stable association is induced by covalent bonding.

[0093] Generally, if the stable association is a non-covalent association, the stable association is a high affinity association. Suitable ligand-reactive moieties may preferably be maleimides, acrylic acid amides (acrylamides), α-haloacetamides, epoxides, O-succinimidyl esters, fluorophenylsulfonamides, and fluorophenylcarboxamides.

[0094] In a further embodiment, the photoswitchable azobiaryl compounds are homofunctional (R 3=R 3 ') or heterofunctional (R 3 ≠R 3 Two ligand-reactive moieties linked in a ') fashion provide an intermolecular bridge to the affinity ligand homodimer or multimer.

[0095] In another embodiment, the photoswitchable azobiaryl compounds are homofunctional (R 3 =R 3 ') or heterofunctional (R 3 ≠R 3 The ligand-reactive moiety linked in an ') fashion provides stable association of the affinity ligand with a solid support, polymer, polypeptide, oligonucleotide, polynucleotide, nucleic acid, carbohydrate, liposome, nanoparticle, cell, biopolymer, biomolecule, or small molecule.

[0096] In some embodiments, the ligand-reactive moiety provides a covalent bond to at least one amino acid side chain in a polypeptide. Binding of the photoswitchable azobiaryl compound to the affinity ligand can be via a tyrosine, tryptophan, serine, threonine, cysteine, histidine, arginine, lysine, aspartic acid, glutamic acid residue, or any standard or non-standard amino acid in a polypeptide that is accessible for reaction with the ligand-reactive moiety of the photoswitchable azobiaryl compound, preferably via a cysteine, histidine, lysine, or methionine residue.

[0097] In one embodiment, for example, when the amino acid to which the photoswitchable azobiaryl compound is linked is a cysteine ​​residue, the ligand reactive moiety can be, for example, an α-haloacetamide, a vinyl sulfone group, a fluorophenylcarboxamide, a fluorophenylsulfonamide, a maleimide, an epoxide, or a substituted maleimide (e.g., NHCOCHCl, NHCOCHBr, NHCOCHI, NH(vinylsulfonate), N(maleimide), N(2-bromomaleimide), N(2,3-dibromomaleimide), NHCOPhF, NHSOPhF, (CH n N(maleimide), (CH2) n N(2-bromomaleimide), (CH2) n N(2,3-dibromomaleimide), NHCO(CH2) n N(maleimide), NHCO(CH2) n N(2-bromomaleimide), NHCO(CH2) n These include groups such as N(2,3-dibromomaleimide, NHCH2CH(O)CH2, N(CH2CH(O)CH2)2)), where n can be any integer from 0 to 10.

[0098] When the amino acid to which the photoswitchable azobiaryl compound is linked is a lysine residue, the ligand-reactive moiety, in some embodiments, comprises a group such as an active ester, e.g., N-hydroxysuccinimidyl ester (generated by N-hydroxysuccinimide and EDC), an epoxide, an isothiocyanate, or an isocyanate.

[0099] Preferably, R 3 and R 3’ is capable of forming an intramolecular bond within the affinity ligand via an amino acid side chain, more preferably comprising a cysteine ​​residue, a histidine residue, a lysine residue, a methionine residue, or any standard or non-standard amino acid in the polypeptide of the affinity ligand that is accessible for reaction with the ligand-reactive moiety of the photoswitchable azobiaryl compound.

[0100] For example, in some embodiments, R 3 and R 3’ is a thiol-reactive moiety covalently attached via an azobenzene core, characterized in that the thiol-reactive moiety contains a reactive electrophile for reaction with the nucleophile of the affinity ligand. In one preferred embodiment, the intramolecular bond within the affinity ligand is in a heterofunctional fashion (R 3 ≠R 3’ In another preferred embodiment, the intramolecular bonds within the affinity ligand are formed in a homofunctional manner (R 3 =R 3’ ) is caused by

[0101] Also preferably, R 3 and R 3’ can form an intermolecular bond between the affinity ligand and the second affinity ligand and / or solid support via an amino acid side chain, more preferably the amino acid side chain involves a cysteine ​​residue, a histidine residue, a lysine residue, a methionine residue, or any standard or non-standard amino acid in a polypeptide or functional group of a solid support that is accessible for reaction with the ligand-reactive moiety of a photoswitchable azobiaryl compound.

[0102] In one preferred embodiment, R 3 and / or R 3’ may be a haloacetamide, more preferably iodoacetamide or chloroacetamide. In one preferred embodiment, R 3 and R 3’ are both hydrogen, then at least one R 2 or R 2’ is not COOH.

[0103] In one preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-iodoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene (alternatively referred to herein as PS1).

[0104] The aforementioned structure PS1 can be depicted as follows:

[0105] [ka]

[0106] In another preferred embodiment of the first aspect of the present invention, the compound is 4'-(2-(4-(2-chloroacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene (alternatively referred to herein as PS2).

[0107] The aforementioned structure PS2 can be depicted as follows:

[0108] [ka]

[0109] In another preferred embodiment of the first aspect of the present invention, the compound is 4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamide)acetic acid)-4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamide)acetic acid)diazene (alternatively referred to herein as PS3).

[0110] The aforementioned structure PS3 can be depicted as follows:

[0111] [ka]

[0112] In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-bromoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-bromoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0113] [ka]

[0114] In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0115] [ka]

[0116] In another preferred embodiment of the first aspect of the present invention, the compound is 4'-(2-(4-(2-chloroacetamido)-3',5'-diethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-diethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0117] [ka]

[0118] In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-iodoacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0119] [ka]

[0120] In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-bromoacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-bromoacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0121] [ka]

[0122] In another preferred embodiment of the first aspect of the present invention, the compound is 4'-(2-(4-(2-chloroacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene (alternatively referred to herein as PS4).

[0123] [ka]

[0124] In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0125] [ka]

[0126] In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0127] [ka]

[0128] In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2-iodoacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0129] [ka]

[0130] In another preferred embodiment of the first aspect of the present invention, the compound is 4'-(2-(4-(2-bromoacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-bromoacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0131] [ka]

[0132] In another preferred embodiment of the first aspect of the present invention, the compound is 4'-(2-(4-(2-chloroacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0133] [ka]

[0134] In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamide)acetic acid)-4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamide)acetic acid)diazene.

[0135] [ka]

[0136] In another preferred embodiment of the first aspect of the invention, the compound is 4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(perfluorophenyl)sulfonamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0137] [ka]

[0138] In another preferred embodiment of the first aspect of the present invention, the compound is 4'-(2-(4-(2-chloroacetamido)-3',5'-dibromobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-dibromobiphenyl-3-ylcarboxamido)acetic acid)diazene.

[0139] [ka]

[0140] In a preferred embodiment of the first aspect of the invention, the compound is more compact in the cis state and more extended in the trans state, more preferably adjacent C 4 The distance between cis and trans atoms differs by about 0.5 nm to about 50 nm, even more preferably by about 1 nm to about 30 nm, and most preferably by about 1 nm to about 20 nm.

[0141] The cis and trans states were modeled via computational chemistry using an energy minimization algorithm (MM2 force field method available in ChemBio3D 19.0 from PerkinElmer Informatics, Inc.). The atomic distances at the p-position of the second aryl were measured in each state using ChemDraw software (ChemDraw Professional Edition 20.1.0.112, PerkinElmer Informatics, Inc.).

[0142] In one preferred embodiment of the first aspect of the present invention, the configuration of the photoswitchable azobiaryl compound can be reversibly altered by irradiation with light of a particular wavelength, more preferably the configuration is switched from a cis to a trans state, or alternatively more preferably the configuration is switched from a trans to a cis state.

[0143] In one preferred embodiment of the preceding embodiment of the first aspect of the present invention, the wavelength of the light is at least 400 nm, more preferably at most 750 nm, and even more preferably at most 700 nm.

[0144] In a preferred embodiment of the first aspect of the present invention, at least 80% of the compounds are in the trans state when exposed to light of a wavelength of about 400 nm to 490 nm (first wavelength λ1) and 80% are in the cis state when exposed to light of a wavelength of about 600 nm to 700 nm (second wavelength λ2).

[0145] In another preferred embodiment of the first aspect of the invention, the compound is soluble in water at pH 8.0 at room temperature from about 0.001 mM to about 2 mM, more preferably greater than 0.1 mM, and most preferably greater than 1 mM.

[0146] In a preferred embodiment of the first aspect of the invention, the thermal half-life of the cis form in water at pH 8.0 at room temperature is from about 1 minute to about 72 hours, more preferably greater than 1 hour, and most preferably greater than 12 hours. The cis to trans ratio was determined by HPLC analysis.

[0147] The photoswitchable azobiaryl compound may change from a first isomeric state to a second isomeric state upon exposure to light of different wavelengths, or upon a change in exposure from dark to light or from light to dark. For example, in some embodiments, the photoswitchable azobiaryl compound may be in a first isomeric state when exposed to light of a first wavelength λ1 and in a second isomeric state when exposed to light of a second wavelength λ2.

[0148] The first wavelength and the second wavelength may differ from each other by about 1 nm to about 1000 nm or more, preferably about 50 nm to about 500 nm, more preferably about 80 nm to about 400 nm, and particularly preferably about 100 nm to about 300 nm. In a preferred embodiment, the first wavelength and the second wavelength differ from each other by at least 100 nm. In another preferred embodiment, the first wavelength and the second wavelength differ from each other by at most 300 nm.

[0149] In other embodiments, the photoswitchable azobiaryl compound is in a first isomeric state when exposed to light of a certain wavelength and in a second isomeric state in the absence of light (e.g., in the absence of light, the photoswitchable azobiaryl compound undergoes thermal relaxation to the second isomeric state). In these embodiments, the first isomeric state is induced by exposure to light of wavelength λ1, and the second isomeric state is induced by not exposing the photoswitchable azobiaryl compound to light, e.g., by keeping the photoswitchable azobiaryl compound in the dark.

[0150] In other embodiments, the photoswitchable azobiaryl compound is in a first isomeric state in the absence of light, e.g., when the photoswitchable azobiaryl compound is in the dark, and the photoswitchable azobiaryl compound is in a second isomeric state when exposed to light of wavelength λ2.

[0151] In other embodiments, the photoswitchable azobiaryl compound is in a first isomeric state when exposed to light of a first wavelength, and the photoswitchable azobiaryl compound is in a second isomeric state when exposed to light of a second wavelength. For example, in some embodiments, the photoswitchable azobiaryl compound is in a trans configuration in the absence of light or when exposed to light of a first wavelength, and the trans configuration is in a cis configuration when exposed to light or when exposed to light of a second wavelength different from the first wavelength.

[0152] As another example, in some embodiments, the photoswitchable azobiaryl compound is in the cis configuration in the absence of light or when exposed to light of a first wavelength, and the photoswitchable azobiaryl compound is in the trans configuration when exposed to light or when exposed to light of a second wavelength that is different from the first wavelength.

[0153] The wavelength of light that effects the change from the first isomeric state to the second isomeric state generally ranges from 1 nm to about 2000 nm. "Light," as used herein, refers to electromagnetic radiation, including, but not limited to, ultraviolet light, visible light, infrared light, and microwaves.

[0154] In some embodiments, the light intensity is about 1 W / m 2 to about 50 W / m 2 Up to, for example, about 1 W / m 2 to about 5W / m 2 Up to approx. 5W / m 2 to about 10 W / m 2 Up to approximately 10W / m 2 From about 10W / m 2 to about 15 W / m 2 Up to approximately 15W / m 2 to about 20 W / m 2 Up to approximately 20W / m 2 to about 30 W / m 2 Up to approximately 30W / m 2 to about 40 W / m 2 up to, or approximately 40W / m 2 to about 50 W / m 2It can vary up to.

[0155] In other embodiments, the light intensity is about 1 μW / cm 2 to approximately 100 μW / cm 2 up to, for example, about 1 μW / cm 2 to approximately 5 μW / cm 2 up to about 5 μW / cm 2 to approximately 10 μW / cm 2 up to about 10 μW / cm 2 to approximately 20 μW / cm 2 up to approximately 20 μW / cm 2 to approximately 25 μW / cm 2 up to approximately 25 μW / cm 2 to approximately 50 μW / cm 2 up to approximately 50 μW / cm 2 to approximately 75 μW / cm 2 up to, or about 75 μW / cm 2 to approximately 100 μW / cm 2 It can vary up to.

[0156] In a further embodiment, the light intensity is about 1 μW / mm 2 to approximately 1 W / mm 2 Up to, for example, about 1 μW / mm 2 to approximately 50 μW / mm 2 up to approximately 50 μW / mm 2 to approximately 100 μW / mm 2 up to approximately 100 μW / mm 2 to approximately 500 μW / mm 2 up to approximately 500 μW / mm 2 to approximately 1 mW / mm 2 up to about 1 mW / mm 2 to approximately 250 mW / mm 2 up to approximately 250 mW / mm 2 to approximately 500 mW / mm 2 up to, or about 500 mW / mm 2 to approximately 1 W / mm 2 It fluctuates up to.

[0157] Also provided in a second aspect of the present invention is a photoswitchable affinity ligand comprising an affinity ligand stably associated with a photoswitchable azobiaryl compound according to the first aspect of the present invention as described above.

[0158] In the context of the present invention, an affinity ligand may be defined as a chemical entity that is preferably capable of interacting with and binding to a target molecule specifically and selectively. In a preferred embodiment of the second aspect of the invention, the affinity ligand is selected from the group consisting of a peptide, oligopeptide, polypeptide, protein, antibody or antigen-binding fragment thereof, immunoglobulin or fragment thereof, enzyme, hormone, cytokine, complex, oligonucleotide, polynucleotide, nucleic acid, aptamer, carbohydrate, liposome, nanoparticle, cell, biopolymer, biomolecule or small molecule.

[0159] In one preferred embodiment of the second aspect of the invention, the affinity ligand is selected from the group comprising immunoglobulin (Ig) binding proteins, more preferably from the group comprising protein A, protein G and protein L, or variants thereof capable of specifically binding to immunoglobulins.

[0160] Protein A, Protein G, or Protein L affinity chromatography is often used in commercial purification processes for pharmaceutical-grade monoclonal antibodies. Protein A is a bacterial cell wall protein that binds to mammalian antibodies primarily through hydrophobic interactions, along with hydrogen bonds and two salt bridges with the Fc region of the antibody.

[0161] Thus, in the context of chromatographic purification, Protein A resins allow affinity-based retention of antibodies on chromatographic supports, while unwanted components in the clarified harvest can flow off the support and be discarded. The retained antibodies can then be eluted from the chromatographic support by disrupting the antibody-Protein A interaction. Typical elution conditions utilizing low pH positively charge the highly conserved (de)ionizable amino acid residues facing each other on the Protein A-Fc region, thus repelling each other and reducing the hydrophobic contact area between the two molecules. However, typical elutions based on acidic elution conditions and low pH levels can lead to chemical modification or denaturation of the antibody and / or affinity matrix, thus affecting functionality.

[0162] The modification of wild-type protein A (SEQ ID NO: 1, UniProtKB entry P38507), wild-type protein G (SEQ ID NO: 2, UniProtKB entry P19909), or wild-type protein L (SEQ ID NO: 3, UniProtKB entry Q51918) with stable association of photoswitchable azobiaryl compounds to optically control their binding activity, and their application to generate photoswitchable affinity matrices, alleviates the disadvantages of this traditional purification technique.

[0163] When coupling affinity ligands to solid supports, multipoint or single-point coupling can be used. Multipoint coupling can prevent three-dimensional structural flexibility and therefore structural rearrangement by modifying the photoswitchable azobiaryl compound. The advantage of single-point coupling is that it allows greater flexibility in the three-dimensional structure of the affinity ligand, potentially preserving its binding properties. This can result in higher specificity and activity compared to multipoint coupling. Single-point coupling can be achieved by covalently coupling the affinity ligand to the solid support via reactive functional groups on the ligand, such as primary amines or thiols, using standard coupling reactions. Using primary amines for covalent immobilization, single-point coupling can be achieved by protein variants with one or a defined set of lysine residues at specific positions, or without lysine residues.

[0164] In one preferred embodiment, the present invention relates to affinity ligands that have a single (N-terminal) or, by introduction of lysine residues, a defined set of amino groups for site-specific single or multipoint attachment.

[0165] In one preferred embodiment, the present invention relates to affinity ligands that are variants of immunoglobulin (Ig)-binding proteins, such as Protein A, Protein G, or Protein L. Variants may include Ig-binding proteins with at least one residue substituted with a cysteine ​​residue. At least one substitution may provide a conjugation site for stable association with a photoswitchable azobiaryl compound bearing one or two reactive sites. Modification of the Ig-binding protein with a photoswitchable azobiaryl compound may provide optical modulation of binding activity.

[0166] Protein A contains five homologous Ig-binding domains that each fold into a three-helix bundle. Each of these five domains is capable of binding to antibodies from many mammalian species, most notably those belonging to the immunoglobulin G (IgG) class. For affinity purification purposes, recombinant fragments of protein A are often used, which comprise or consist of domain B of protein A. More specifically, protein A binds to the Fc region within the heavy chains of most immunoglobulins, and also to the Fab region, particularly in the case of the human VH3 family.

[0167] In one preferred embodiment of the second aspect of the invention, the affinity ligand comprises the B domain of protein A (SEQ ID NO: 4), optionally substituted with one or two cysteine ​​residues, preferably comprising residues 215-268 of SEQ ID NO: 1 (UniProtKB entry P38507, amino acid numbering based on the full length sequence), or a protein domain having at least 80% sequence identity to SEQ ID NO: 4; more preferably, the affinity ligand comprises a protein domain having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7; even more preferably, the affinity ligand comprises a protein domain having the amino acid sequence of SEQ ID NO: 7.

[0168] In another preferred embodiment of the second aspect of the present invention, the affinity ligand comprises the lysine-poor B domain of protein A (SEQ ID NO: 5), optionally substituted with one or two cysteine ​​residues, or comprises a protein domain having at least 80% sequence identity with SEQ ID NO: 5; more preferably, the affinity ligand comprises a protein domain having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11; even more preferably, the affinity ligand comprises a protein domain having the amino acid sequence of SEQ ID NO: 11.

[0169] In one preferred embodiment, the affinity ligand may comprise the wild-type (SEQ ID NO: 4) or lysine-deficient B domain (SEQ ID NO: 5) of protein A. According to another preferred embodiment, the wild-type or lysine-deficient B domain of protein A may be mutated to have one or more amino acid substitutions or mutations of residues selected from the group including Lys215, Phe216, Asn217, Lys218, Glu219, Asn234, Glu236, Gly240, Phe241, Lys246, Asp247, Asp248, Ser250, Ala253, Asn254, Lys260, Lys261, Ala265 and Ala267 (shown in bold in SEQ ID NO: 4, as presented below), and may be used.

[0170] In one preferred embodiment, the single substitution may preferably be selected from Asn217Cys (i.e. Asn at position 217 substituted or mutated with cysteine), Glu219Cys, Glu236Cys, Gly240Cys, Asp247Cys, Asp248Cys, Ser250Cys, Ala253Cys, Asn254Cys, Ala265Cys or Ala267Cys. The double substitution may preferably be selected from Asn217Cys / Asp248Cys, Glu219Cys / Asp248Cys, Glu219Cys / Ala267Cys Glu236Cys / Asp247Cys, Gly240Cys / Ala265Cys or Gly240Cys / Asn254Cys.

[0171] In another preferred embodiment, residues 215-216 in SEQ ID NO: 4 or SEQ ID NO: 5 may preferably be replaced by Lys213-Ala214-Cys215-Gly216 (as in SEQ ID NOs: 6 to 9), each preferably having one additional substitution selected from Asp247Cys (SEQ ID NO: 7), Asp248Cys, Ser250Cys (SEQ ID NO: 9) or Ala253Cys.

[0172] In yet another embodiment, residues 215-216 in SEQ ID NO: 5 may preferably be replaced by Lys209-Gly210-Gly211-Gly212-Gly213-Ala214-Ser215-Phe216 to provide a linker between the solid support and the affinity ligand, preferably with a double substitution selected from Asn217Cys / Asp248Cys (SEQ ID NO: 10), Glu219Cys / Asp248Cys (SEQ ID NO: 11), Glu219Cys / Ala267Cys Glu236Cys / Asp247Cys, Gly240Cys / Ala265Cys or Gly240Cys / Asn254Cys.

[0173] The particular sequence of the Ig-binding affinity ligand may preferably be extended at the N-terminus with a Met residue and at the C-terminus with Ser-Ala-His-His-His-His-His-His-His.

[0174] [ka]

[0175] Protein G is also an immunoglobulin-binding protein found in group C and G streptococci. It consists of three Ig-binding domains with specific binding affinity for antibody Fc and Fab regions, as well as an albumin-binding region.

[0176] In one embodiment of the second aspect of the invention, the affinity ligands of the invention may comprise at least one of the three homologous domains of protein G in SEQ ID NO: 2 (UniProtKB entry P19909; amino acid numbering is based on the full-length sequence), preferably defined as C1 (residues 303-357; also referred to elsewhere as B1), C2 (residues 373-427) and C3 (residues 443-497; also denoted B2).

[0177] In one embodiment of the second aspect, the affinity ligand of the present invention may preferably comprise a domain of streptococcal protein G, and more preferably may have at least one residue of the C1, C2 or C3 domain of protein G substituted with a cysteine ​​residue.

[0178] In another preferred embodiment of the second aspect of the invention, the affinity ligand comprises at least one of the three homologous domains of protein G defined as C1 (SEQ ID NO: 12), C2 (SEQ ID NO: 13) and C3 (SEQ ID NO: 14), which may have two substitutions of wild-type residues by cysteines, or comprises a protein domain having at least 80% sequence identity with SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.

[0179] In another preferred embodiment of the second aspect of the invention, the affinity ligand of the invention comprises the lysine-deficient C1 domain of protein G with an Asn338Tyr substitution, in which the lysine residue is substituted with any other amino acid except lysine, to increase stability against alkaline hydrolysis (SEQ ID NO: 15).

[0180] Each domain may be extended at the N-terminus with the sequence Met-Lys (SEQ ID NOs: 12, 13, and 14) or Met-Ser (SEQ ID NO: 15), and each domain may have single or double substitutions, such as mutations, of the corresponding residues. More specifically, the protein G domain C1 used herein preferably contains one or more substitutions of residues Thr303, Lys305, Lys314, Lys311, Glu316, Lys314, Glu320, Val322, Lys329, Lys332, Asp337, Asn338, Thr345, Asp348, Lys351, Thr350, and Thr356 (shown in bold in SEQ ID NO: 12 below). Accordingly, one or more equivalent substitutions may preferably be included at the corresponding homologous positions in domains C2 and C3. These substitutions are also considered preferred, either individually or in all possible combinations.

[0181] The single substitution within domain C1 may preferably be selected from Thr303Cys (i.e., Thr at position 303 substituted or mutated with cysteine), Lys305Cys, Lys311Cys, Lys314Cys, Glu316Cys, Glu320Cys, Val322Cys, Lys329Cys, Lys332Cys, Asp337Cys, Asn338Cys, Thr345Cys, Asp348Cys, Thr350Cys, Lys351Cys, and Thr356Cys.

[0182] The double substitution within domain C1 may preferably be selected from Thr303Cys / Thr356Cys, Lys305Cys / Lys314Cys, Lys305Cys / Thr445Cys, Lys305Cys / Thr356Cys, Lys311Cys / Thr350Cys, Lys314Cys / Thr350Cys, Lys314Cys / Thr356Cys, Glu316Cys / Thr345Cys, Glu320Cys / Asp337Cys, Glu320Cys / Asn338Cys, Glu320Cys / Thr350Cys, Val322Cys / Asp348Cys or Thr350Cys / Thr356Cys.

[0183] Domains C2 and C3 may preferably contain single and double Cys mutations at the corresponding homologous positions, and each sequence may be extended at the C-terminus with the sequence Ser-Ala-His-His-His-His-His-His.

[0184] [ka]

[0185] Protein L is a bacterial surface protein important for pathogenic immune evasion. The full-length protein L of Finegoldia magna contains an N-terminal region with affinity for a diverse set of immunoglobulins, an additional region with an albumin-binding domain, and cell wall-spanning and membrane-anchoring domains. Four homologous Ig-binding domains found in Finegoldia magna strain 3316 have specific affinity for immunoglobulin light chains.

[0186] From the standpoint of antibody purification, interaction with the (kappa) light chain is an advantage over protein A or G because additional antibody classes such as IgA, IgM, IgE, or IgD can be bound by protein L. Furthermore, the light chain specificity of the Ig-binding affinity ligands allows for the purification of antibody fragments such as single-chain variable fragments (scFv) and Fab fragments or fusions thereof.

[0187] In one preferred embodiment of the second aspect of the present invention, the affinity ligand comprises one C domain of protein L having at least one residue substituted with a cysteine ​​residue, or a protein domain having at least 80% sequence identity thereto; more preferably, the C domain of protein L is the C2 domain of protein L (SEQ ID NO: 16), defined as residues 326 to 389 corresponding to UniProtKB entry Q51918 (SEQ ID NO: 3), or a protein domain having at least 80% sequence identity thereto.

[0188] In another preferred embodiment of the second aspect of the invention, the affinity ligand of the invention comprises the C2 domain of protein L (SEQ ID NO: 17) in which the lysine residues have been replaced with any other amino acid except lysine, providing site-specific immobilization via amino-reactive chemistry.

[0189] In one preferred embodiment, the Ig-binding affinity ligand of the present invention may comprise the C2 domain of protein L (SEQ ID NO: 16). More specifically, protein L domain C2 as used herein preferably comprises one or more substitutions of residues Lys326, Lys332, Ile336, Lys341, Thr342, Lys348 Glu353, Lys357, Lys367, Glu377, Asp378, Thr382 and Lys386 (shown in bold in SEQ ID NO: 16 depicted below).

[0190] Single substitutions may be selected from Ile336Cys (i.e., Ile substituted or mutated at position 336 with cysteine), Thr342Cys, Glu353Cys, Lys367Cys, Glu377Cys, or Asp378Cys. Double substitutions may be selected from Ile336Cys / Asp378Cys, Thr342Cys / Glu377Cys, or Thr342Cys / Asp378Cys. The specific sequence of the Ig-binding affinity ligand may be extended at the N-terminus with a Met residue and at the C-terminus by the sequence His-His-His-His-His-His.

[0191] [ka]

[0192] Below, the amino acid sequences of specific preferred individual affinity ligand protein domains are given in common single letter order. SEQ ID NO: 4 (B domain of protein A) MKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAP SEQ ID NO: 5 (Lysine-poor B domain of protein A) MSFNMEQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEAQELNDAQAP SEQ ID NO: 6 (affinity ligand protein domain) MKACGNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAP SEQ ID NO: 7 (affinity ligand protein domain) MKACGNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPCQSANLLAEAKKLNDAQAP SEQ ID NO: 8 (affinity ligand protein domain) MKACGNMEQQNAFYEILHLPNLNEEQRNGFIQSLRDDDPSQSANLLAEAQELNDAQAP SEQ ID NO: 9 (affinity ligand protein domain) MKACGNMEQQNAFYEILHLPNLNEEQRNGFIQSLRDDPCQSANLLAEAQELNDAQAP SEQ ID NO: 10 (affinity ligand protein domain) MKGGGGASFCMEQQNAFYEILHLPNLNEEQRNGFIQSLRDCPSQSANLLAEAQELNDAQAP SEQ ID NO: 11 (affinity ligand protein domain) MKGGGGASFNMCQQNAFYEILHLPNLNEEQRNGFIQSLRDCPSQSANLLAEAQELNDAQAP SEQ ID NO: 12 (protein G domain C1, residues 303-357 of SEQ ID NO: 2) MKTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE SEQ ID NO: 13 (protein G domain C2, residues 373-427 of SEQ ID NO: 2) MKTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE SEQ ID NO: 14 (protein G domain C3, residues 443-497 of SEQ ID NO: 2) MKTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE SEQ ID NO: 15 (C1 domain of protein G) MSTYRLILNGVTLSGETTTEAVDAATAERVFRQYANDYGVDGEWTYDDATRTFTVTE SEQ ID NO: 16 (C2 domain of protein L) MKEEVTIKVNLIFADGKTQTAEFKGTFEEATAKAYAYADLLAKENGEYTADLEDGGNTINIKFAG SEQ ID NO: 17 (Lysine-poor C2 domain of protein L) MAEEVTIRVNLIFADGSTQTAEFRGTFEEATAEAYAYADLLARENGEYTADLEDGGNTINIRFAG SEQ ID NO: 18 (Variant SpG#1 of the C1 domain of protein G) MATKASKGGGGTTYRLILNGVTLSGETTTEACDAATAERVFRQYANDYGVDGEWTYDCATRTFTVTEGEDEDEDED SEQ ID NO: 19 (Variant PpL#1 of the C2 domain of protein L) MATKASKGGGGASEEVTIRVNLIFADGSTQCAEFRGTFEEATAEAYAYADLLARENGEYTACLEDGGNTINIRFAGEDEDEDED In one preferred embodiment of the second aspect of the present invention, the protein L domain C2 used herein preferably comprises one or more amino acid substitutions selected from the list consisting of Thr344Cys, Glu346Cys, and Asp375Cys. According to a preferred embodiment, the single substitution may be selected from Thr344Cys, Glu346Cys, and Asp375Cys. Preferably, the double substitution may be selected from Glu346Cys / Asp378Cys and Thr344Cys / Asp375Cys.

[0193] In one preferred embodiment of the second aspect of the present invention, the photoswitchable azobiaryl compound is stably associated with two conjugation sites within the affinity ligand in a bifunctional or bifunctional manner.

[0194] In another preferred embodiment of the second aspect of the invention, exposure to light of a specific wavelength induces a conformational switch, causing a change in the specific affinity of the photoswitchable affinity ligand for the target molecule.

[0195] As discussed above, the present invention provides a photoswitchable affinity ligand, which comprises at least one reactive moiety for stable association with a photoswitchable azobiaryl compound according to the present invention. The affinity ligand may further comprise an additional moiety for stable association with a solid support.

[0196] Modification of affinity ligands can be preferably achieved using mono-, bi-, or bifunctional photoswitchable azobiaryl compounds with respect to the ligand-reactive moiety. Optical modulation of ligand affinity using monofunctional photoswitches relies on steric effects (e.g., interference with ligand binding), whereas modulation using bifunctional photoswitches is usually intended to modulate the conformation of the ligand and hence its specific affinity.

[0197] One preferred embodiment of the present invention relates to affinity ligands that are stably associated in cis with bifunctional or bifunctional photoswitchable azobiaryl compounds. This modification of the affinity ligand is preferably achieved by introducing two conjugation sites (e.g., thiol groups on cysteine ​​side chains), which can be crosslinked in a highly specific manner by the bifunctional photoswitchable azobiaryl compounds. These photoswitchable affinity ligands can be triggered by light illumination to reversibly change their conformation and / or their affinity.

[0198] Photoswitchable azobiaryl compounds conjugated to affinity ligands at two specific positions (conjugation sites) in a more compact cis state are expected to preserve the native conformation of the affinity ligand upon exposure to a first wavelength of light. Photoisomerization from the more compact cis state to the elongated trans state upon exposure to a second wavelength of light preferably distorts the affinity ligand, thus impairing binding of the target molecule.

[0199] Another preferred embodiment relates to the modification of affinity ligands with bifunctional or bifunctional photoswitches in the trans state. Photoswitchable azobiaryl compounds bound in the trans state, extending at two specific positions (conjugation sites), are expected to preserve the natural conformation of the affinity ligand upon exposure to light of a first wavelength. Photoisomerization from the trans state to a more compact cis state upon exposure to light of a second wavelength preferably distorts the affinity ligand, thus impairing binding of the target molecule.

[0200] Another preferred embodiment relates to the modification of affinity ligands with monofunctional photoswitches in the cis state: a photoswitchable azobiaryl compound attached at one specific position (conjugation site) preferably allows binding of the target molecule in a more compact cis state upon exposure to light of a first wavelength, but sterically overlaps in an elongated trans state upon exposure to light of a second wavelength, thus impairing binding.

[0201] A further preferred embodiment relates to the modification of affinity ligands with monofunctional photoswitches in the trans state: Photoswitchable azobiaryl compounds attached at one specific position (conjugation site) preferably allow binding of target molecules in the extended trans state upon exposure to light of a first wavelength, but sterically overlap in the more compact cis state upon exposure to light of a second wavelength, thus impairing binding.

[0202] In one preferred embodiment, the photoswitchable affinity ligands of the present invention may be modified by recombinant means to include a spacer or linker sequence as either an N- or C-terminal extension, thereby forming a fusion ligand-linker product, which may confer improved immobilization to a solid support. In this regard, as will be appreciated by those skilled in the art, a linker or spacer may be chemically synthesized and covalently attached to a selected photoswitchable affinity ligand using well-established methodologies.

[0203] Alternatively, a fusion product comprising a linker and a photoswitchable affinity ligand of the present invention can be produced using recombinant techniques. In this regard, the linker can generally be attached or fused to a terminus of the photoswitchable affinity ligand, so that the function of the photoswitchable affinity ligand can be substantially retained. Thus, depending on the protein, the linker can be attached to the N-terminus or C-terminus of the photoswitchable affinity ligand. In certain cases, the linker can be attached to both termini or to an additional amino acid residue that is not the terminal residue.

[0204] In another alternative, the linker may be attached to the solid support by chemical means or through the use of an enzyme prior to coupling of the photoswitchable affinity ligand. The linker may be suitable for immobilizing the photoswitchable affinity ligand of the invention on the solid support while substantially retaining the function of the photoswitchable affinity ligand, i.e., while retaining at least about 50% of the immunoglobulin binding in its immobilized state compared to the unbound state.

[0205] Photoswitchable affinity ligands according to the present invention can be prepared by selecting at least one conjugation site within the affinity ligand for stable association of the photoswitchable azobiaryl compound. The conjugation site, such as a cysteine ​​residue, may be added to the affinity ligand by substitution or insertion if not already present. The conjugation site may be a ligand-reactive moiety or a group R as shown in formula (I). 3 / R 3 The functional moiety must be suitable for conjugation with additional functional moieties as described herein as '.

[0206] Photoswitches can be covalently associated with selected conjugation sites through an assortment of different conjugation chemistries, as described herein and known in the art. For example, a photoswitchable azobiaryl compound bearing a reactive iodoacetyl group that targets two accessible cysteine ​​thiols on a polypeptide is one embodiment, although numerous conjugation or coupling chemistries targeting the side chains of either standard or non-standard amino acids can be used in accordance with the present invention.

[0207] The selection of the location of the conjugation site on the affinity ligand is another important aspect. Any exposed amino acid residue on the affinity ligand surface can be a potentially useful conjugation site and, if not already present at the selected conjugation site in the affinity ligand sequence, can be mutated to cysteine ​​or some other reactive amino acid for covalent association. Steric hindrance between the crosslinked photoswitch and the affinity ligand binding cleft should be avoided so that specific binding is preserved.

[0208] In one preferred embodiment, a water-soluble derivative of a photoswitch according to the present invention is selected to stabilize the native conformation of an affinity ligand (e.g., protein A) in a more compact cis state and to distort it into an extended trans state. This criterion determines the distance between conjugation sites within the affinity ligand, which are crosslinked via a bifunctional (thiol-reactive) photoswitch. In this regard, the ideal distance between conjugation sites can be inferred from the length of the photoswitchable azobiaryl compound in its cis state, approximately 11-16 Å (Figure 1D). Using the protein structure (model) of the affinity ligand, suitable residues (cysteine ​​pair; Sγ side chain atom) that satisfy this distance criterion can be determined.

[0209] In a further preferred embodiment, the water-soluble derivative of the photoswitch according to the invention is selected to stabilize the native conformation of the affinity ligand in the trans state and to destabilize it in the more compact cis state, this criterion determining the distance between the conjugation sites that are bridged via the bifunctional (thiol-reactive) photoswitch.

[0210] For this purpose, the ideal distance between conjugation sites can be estimated from the length of the photoswitchable azobiaryl compound in its trans state, approximately 20-26 Å (Figure 1C). Using the protein structure (model) of the affinity ligand, suitable residues (cysteine ​​pair; Sγ side chain atom) that satisfy this distance criterion can be determined.

[0211] According to a third aspect of the present invention, there is provided a photoswitchable affinity matrix comprising a solid support and a photoswitchable azobiaryl compound according to the first aspect of the present invention in stable association with an affinity ligand, wherein the photoswitchable azobiaryl compound and affinity ligand are in stable association with the solid support; or a photoswitchable affinity matrix comprising a photoswitchable affinity ligand according to the second aspect of the present invention in stable association with the solid support.

[0212] The photoswitchable matrix according to the present invention is preferably provided for optically controlled affinity separation. The matrix may comprise a photoswitchable affinity ligand, which may be made from any of the affinity ligands of the present invention and a photoswitchable azobiaryl compound coupled to a solid support. The affinity matrix according to an embodiment of the present invention exhibits isolation of a target from an aqueous mixture under the control of light.

[0213] The affinity ligand can be coupled (covalently or non-covalently) to the solid support, preferably before or after it is functionalized via the stable association of the photoswitchable azobiaryl compound. In one preferred embodiment, the coupling of the photoswitchable affinity ligand to the solid support is mediated between the affinity ligand moiety and the solid support. In another preferred embodiment, the coupling of the photoswitchable affinity ligand to the solid support is mediated between the photoswitchable azobiaryl compound moiety and the solid support. In yet another preferred embodiment, both the affinity ligand moiety and the photoswitchable azobiaryl compound moiety contribute to the coupling of the photoswitchable affinity ligand to the solid support. Preferably, the coupling comprises a covalent bond.

[0214] The (photoswitchable) affinity ligands of the present invention may be attached to a solid support via conventional coupling techniques utilizing, for example, amino and / or carboxy groups present in the ligand or any other functional groups of the affinity ligand and / or the photoswitchable azobiaryl compound. The use of epoxide-, CNBr-, N-hydroxysuccinimidyl ester-activated solid supports and solid supports for copper-catalyzed click chemistry are well-known immobilization procedures.

[0215] A spacer or linker may be introduced between the support and the ligand to facilitate chemical coupling of the affinity ligand to the support, which improves the availability of the photoswitchable affinity ligand. Alternatively, the photoswitchable affinity ligand may be bound to the support by non-covalent association, such as physical or biospecific adsorption.

[0216] In one embodiment, the (photoswitchable) affinity ligands of the present invention may be coupled to the support via a primary amine (e.g., a lysine side chain or the N-terminus). Methods for performing such attachment are well known in the art and are readily performed by those skilled in the art using standard techniques and equipment.

[0217] Suitable solid supports are preferably selected from the group comprising synthetic polymers (e.g., polysulfone (PSF), polyethersulfone (PES), polyacrylonitrile (PAN), polyamide (PA), polyethylene and polypropylene (PE and PP), polymethyl methacrylate (PMMA), polyglycidyl methacrylate (PGMA), polysterene (PS)), non-synthetic polymers, for example polysaccharides (e.g., dextran, starch, cellulose, pullulan, or agarose), inorganic supports (e.g., silica or zirconium oxide, magnetic particles), and any mixed composite solid supports derived from the mentioned or any surface with chemistry allowing the covalent association (chemical coupling) of affinity ligands and / or photoswitches.

[0218] Examples of solid support materials are based on polymers with surface chemistries for covalent association, such as, but not limited to, polymers with hydroxyl groups (-OH), carboxyl groups (-COOH), amino groups (-NH, possibly in substituted form), epoxide groups, azide or alkyne groups for click chemistry.

[0219] In one preferred embodiment, the polymer is a synthetic polymer (e.g., polyethersulfone). Such synthetic polymers may be commercially available products. In another preferred embodiment, the polymer is a polysaccharide (e.g., dextran, starch, cellulose, pullulan, or agarose). Such polysaccharides may be commercially available products.

[0220] In another preferred embodiment, the solid support is a magnetic particle. Such magnetic particles may be commercially available products. The solid support may preferably be in the form of particles. The particles may be porous or non-porous. The solid support in the form of particles may be used as a packed bed or in a suspended form. The suspended form may be an expanded bed or a pure suspension, in which the particles can move freely. When using a packed bed or an expanded bed, separation processes used in known affinity chromatography methods may be used. When using a pure suspension, a batch method may be used.

[0221] Solid supports in the form of particles according to this embodiment may preferably have a particle size (diameter) of from about 1 nm to about 500 micrometers, more preferably from about 100 nm to about 100 micrometers. Particle size may be determined by light scattering, preferably using a suitable particle size analyzer manufactured by Malvern Panalytical.

[0222] In alternative embodiments, the solid support may be in another form, such as a monolith, a chip, a microtiter plate, a capillary tube, or a membrane. In a preferred embodiment, the solid support may be in the form of a membrane. According to a preferred embodiment of the present invention, a porous membrane with a large internal surface area is used. 3 2 to 300m per 2 A porous membrane having a BET surface area of ​​1 cm is preferred. 3 8 to 30 m per 2Even more preferred are those membranes having a BET surface area of ​​0.05 to 0.05 mm. The BET method for determining the surface area of ​​porous membrane structures is based on nitrogen adsorption measurements and is described by K. Kaneko (Kaneko, K. (1994). Determination of pore size and pore size distribution: 1. Adsorbents and catalysts. Journal of membrane science, 96(1-2), 59-89.).

[0223] There are no limitations regarding the material of the membrane according to the invention. Membranes made of inorganic materials such as glass, ceramic, SiO2, carbon, or metal, or of organic polymers or blends thereof, can be used. The polymers can be hydrophilic and / or hydrophobic in nature.

[0224] They may be selected from the group of cellulose polymers, such as cellulose or regenerated cellulose, modified celluloses, such as cellulose esters, cellulose ethers, amine-modified cellulose, or blends of cellulose polymers, synthetic polymers, such as polyacrylonitrile and corresponding copolymers, polymers containing polyurethanes, polyarylsulfones and polyarylethersulfones, such as polysulfones or polyethersulfones, polyvinylidene fluoride, polyacrylamide, polytetrafluoroethylene, water-insoluble polyvinyl alcohols, aliphatic and aromatic polyamides, polyimides, polyetherimides, polyesters, polycarbonates, polyolefins, such as polyethylene, polypropylene, polyvinyl chloride, polyphenylene oxide, polybenzimidazole and polybenzimidazolone, as well as modifications, blends, mixtures, or copolymers derived from these polymers.

[0225] Other polymers, such as polyethylene oxide, polyhydroxyether, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, or polycaprolactone, or inorganic materials such as SiO2, may be mixed with these polymers or polymer blends as additives. In individual cases, the membrane may be subjected to surface modification to establish certain properties of the membrane surface, such as the formation of certain functional groups. When using polyolefin polymers, it may be necessary to coat at least the inner surface of the membrane with a polymer that allows functionalization.

[0226] According to a fourth aspect of the present invention there is provided the use of a photoswitchable compound for isolating and / or purifying a target molecule. Interestingly, there is a lack of sufficient or enabling disclosure of the use of photoswitchable compounds for isolating and / or purifying target molecules as disclosed and described herein. Thus, the inventors and applicants deserve recognition for the first enabling, implementable, and scalable solution to the problem of providing the use of photoswitchable compounds for isolating and / or purifying target molecules from aqueous solutions containing mixtures of different components.

[0227] In a preferred embodiment of the fourth aspect of the present invention, the compound is not 4'-carboxyphenylazophenylalanine, more preferably the compound is not 3'-carboxyphenylazophenylalanine or 4'-carboxyphenylazophenylalanine, and even more preferably the compound is not 3'-carboxyphenylazophenylalanine or a derivative thereof, or 4'-carboxyphenylazophenylalanine or a derivative thereof.

[0228] In another preferred embodiment of the fourth aspect of the present invention, the photoswitchable compound is a photoswitchable azobiaryl compound comprising at least two substituted biphenyl moieties. In another preferred embodiment of the fourth aspect of the invention, the photoswitchable compound is a photoswitchable azobiaryl compound according to the first aspect of the invention.

[0229] According to a fifth aspect of the present invention there is provided the use of a photoswitchable affinity ligand, comprising an affinity ligand in stable association with a photoswitchable compound, for isolating and / or purifying a target molecule.

[0230] In a preferred embodiment of the fifth aspect of the present invention, the photoswitchable compound is not 4'-carboxyphenylazophenylalanine, more preferably the compound is not 3'-carboxyphenylazophenylalanine or 4'-carboxyphenylazophenylalanine, even more preferably the compound is not 3'-carboxyphenylazophenylalanine or a derivative thereof or 4'-carboxyphenylazophenylalanine or a derivative thereof.

[0231] In another preferred embodiment of the fifth aspect of the invention, the photoswitchable affinity ligand comprises an affinity ligand in stable association with a photoswitchable azobiaryl compound comprising at least two substituted biphenyl moieties.

[0232] In a preferred embodiment of the fifth aspect of the invention, the photoswitchable affinity ligand is a photoswitchable affinity ligand according to the first aspect of the invention. According to a sixth aspect of the present invention, there is provided the use of a photoswitchable affinity matrix comprising a solid support and a photoswitchable compound in stable association with an affinity ligand, wherein the photoswitchable compound and affinity ligand are in stable association with the solid support, or the photoswitchable affinity ligand in stable association with the solid support, for isolating and / or purifying a target molecule.

[0233] Again, such uses have not been disclosed in the prior art and have not been successfully or enablingly identified and disclosed prior to the filing date of this application. In a preferred embodiment of the fifth or sixth aspect of the invention, the photoswitchable compound is not 4'-carboxyphenylazophenylalanine, more preferably the compound is not 3'-carboxyphenylazophenylalanine or 4'-carboxyphenylazophenylalanine, even more preferably the compound is not 3'-carboxyphenylazophenylalanine or a derivative thereof or 4'-carboxyphenylazophenylalanine or a derivative thereof.

[0234] In a preferred embodiment of the fifth or sixth aspect of the invention, the photoswitchable affinity ligand is a photoswitchable affinity ligand according to the second aspect of the invention. In a preferred embodiment of the sixth aspect of the invention, the photoswitchable affinity matrix is ​​a photoswitchable affinity matrix according to the third aspect of the invention.

[0235] In a preferred embodiment of the fourth, fifth or sixth aspect of the invention, the target molecule is an immunoglobulin, more preferably the target molecule is an IgG type immunoglobulin, even more preferably an IgG fragment or modality thereof (e.g. Fab, diabody (dAb), single chain fragment variable region (scFv), bispecific scFv (Bis-scFv), ScFv-Fab, Fc-modified full length IgG, dual affinity retargeting antibody (DART)) or a fusion protein comprising a molecule as previously listed.

[0236] According to a seventh aspect of the present invention, there is provided a method for isolating and / or purifying a target molecule, the method comprising the steps of: providing a composition comprising the target molecule; contacting the composition with an affinity matrix for a time sufficient to allow specific binding of the target molecule to the affinity matrix, the affinity matrix comprising a photoswitchable compound stably associated with an affinity ligand and a solid support to form an affinity matrix, wherein preferably the compound is not 3'-carboxyphenylazophenylalanine or 4'-carboxyphenylazophenylalanine; washing the affinity matrix with a wash solution to remove components of the composition that are not specifically bound to the affinity matrix; irradiating the affinity matrix with light of a wavelength of at least about 400 nm to cause loss of specific binding or affinity of the affinity matrix to the target molecule; and eluting the target molecule from the affinity matrix using an eluent.

[0237] In a preferred embodiment of the seventh aspect of the present invention, the irradiation in step d) is carried out at a wavelength in the range of about 400 nm to about 750 nm, preferably about 400 nm to about 700 nm.

[0238] According to an eighth aspect of the present invention, there is provided a method for isolating and / or purifying a target molecule, the method comprising the steps of: providing a composition comprising the target molecule; contacting the composition with an affinity matrix for a time sufficient to allow specific binding of the target molecule to the affinity matrix, the affinity matrix comprising a photoswitchable azobiaryl compound comprising at least two substituted biphenyl moieties, preferably a photoswitchable azobiaryl compound according to the first aspect of the present invention in stable association with an affinity ligand, wherein the photoswitchable azobiaryl compound and the affinity ligand comprise a photoswitchable azobiaryl compound in stable association with a solid support to form an affinity matrix, or a photoswitchable affinity ligand according to the second aspect of the present invention in stable association with a solid support to form an affinity matrix, or a photoswitchable affinity matrix according to the third aspect of the present invention; washing the affinity matrix with a wash solution to remove components of the composition that are not specifically bound to the affinity matrix; irradiating the affinity matrix with light of a particular wavelength to cause the affinity matrix to lose its specific binding or affinity for the target molecule; and eluting the target molecule from the affinity matrix using an eluent.

[0239] In a preferred embodiment of the eighth aspect of the present invention, the irradiation in step d) is carried out at a wavelength of at least about 400 nm, for example from about 400 nm to about 750 nm, preferably in the range of from about 400 nm to about 700 nm.

[0240] According to a further aspect of the present invention, there is provided a method for isolating and / or purifying a target molecule, the method comprising the steps of: providing a composition comprising the target molecule; contacting the composition with an affinity matrix for a time sufficient to allow specific binding of the target molecule to the affinity matrix, the affinity matrix comprising a photoswitchable compound stably associated with an affinity ligand and a solid support to form an affinity matrix, wherein preferably the compound is not 3'-carboxyphenylazophenylalanine or 4'-carboxyphenylazophenylalanine; washing the affinity matrix with a wash solution to remove components of the composition that are not specifically bound to the affinity matrix; irradiating the affinity matrix with light of a particular wavelength to cause loss of specific binding or affinity of the affinity matrix for the target molecule; and eluting the target molecule from the affinity matrix using an eluent.

[0241] All embodiments and aspects as described and / or claimed herein are contemplated as combinable in any combination within the present invention, unless one skilled in the art would consider such combinations to be without any technical significance or excluded by inconsistencies. [Example]

[0242] Example 1 Synthesis of 4'-(2-(4-(2-iodoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene (PS1) Unless otherwise stated, all chemicals and reagents were available from common suppliers (Carbolution, Sigma Aldrich, Alfa Aesar, Thermo Scientific, VWR). NMR spectra were recorded on a Bruker Avance 500 spectrometer and an Avance 500 spectrometer using QNP-Cryo. Coupling constants are reported in Hz. Compounds were identified from a set of 1D and 2D NMR experiments. 1 H and 13 The compounds were characterized by C resonance. ESI-MS analysis was performed on a Thermo Scientific LCQ-Fleet mass spectrometer coupled to a Thermo Scientific Dionex Ultimate 3000 HPLC system.

[0243] Step 1: Synthesis of 2-(tert-butoxycarbonylamino)-5-iodobenzoic acid (1)

[0244] [ka]

[0245] 26.3 g (100 mmol, 1 equiv.) of 5-iodoanthranilic acid was dissolved in 100 ml of 1 M aqueous NaOH, and the pH was adjusted to 7-8 with additional 1 M aqueous NaOH (compared to universal test paper). 27.8 ml (130 mmol, 1.3 equiv.) of BocO was added, and the mixture was vigorously stirred at room temperature for 8 h. The pH was again adjusted to 7-8 as before. An additional 27.8 ml (130 mmol, 1.3 equiv.) of BocO was added, and the mixture was vigorously stirred at room temperature for 16 h. The mixture was diluted with 300 ml of HO, cooled in an ice bath, and the pH was adjusted to 3-4 with 1 M aqueous HCl. After stirring the suspension at 0 °C for 30 min, the product was collected by vacuum filtration and washed with three portions of HO. After air drying to constant weight, 34.9 g (96 mmol, 96%) of a slightly beige solid was obtained.

[0246]

number

[0247] ESI-MS m / z (accurate mass): Calculated value: 245.94[M-Boc-OH] + , 263.95[M-Boc+H] + , 363.00[M+H] + , 1127.95[3M+K] + ; Actual values: 245.96, 263.77, 362.00, 1127.96 Step 2: Synthesis of methyl 2-(2-(tert-butoxycarbonylamino)-5-iodobenzamido)acetate (2)

[0248] [ka]

[0249] To 2.542 g (7 mmol, 1 equiv.) of N-Boc-5-iodoanthranilic acid (1) and 0.967 g (7.7 mmol, 1.1 equiv.) of HCl·H-Gly-OMe, 4.17 ml (24.5 mmol, 3.5 equiv.) of DIPEA and 7 ml of DMF were added. The mixture was stirred for 3 min, followed by the addition of 4.007 g (7.7 mmol, 1.1 equiv.) of PyBOP. After complete dissolution of the solids, the reaction was stirred at room temperature for 1 h. 35 ml of saturated aqueous NaHCO3 was added, and the mixture was stirred for 5 min. The mixture was extracted with 70 ml of DCM, and the organic phase was washed once with 35 ml of saturated aqueous NaHCO, twice with 35 ml of 0.5 M aqueous HCl, and once with 35 ml of brine, dried over MgSO, filtered, and evaporated under vacuum to give 5.67 g of crude product as a clear yellow oil. The product was purified by column chromatography (SiO, ethyl acetate / cyclohexane, step gradient 5%, 10%, 15%, and 20%). The product-containing fractions were combined and evaporated under reduced pressure to give 2.559 g (5.90 mmol, 84%).

[0250]

number

[0251] ESI-MS m / z (accurate mass): Calculated value: 245.94 [M-Boc-Gly] + , 334.99[M-Boc+H] + , 457.02 [M+Na] + , 891.06 [2M+Na] + ;Actual values: 246.03, 334.84, 456.76, 890.99.

[0252] Step 3: Synthesis of methyl 2-(2-(tert-butoxycarbonylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamido)acetate (3)

[0253] [ka]

[0254] The following reaction steps were carried out under an N atmosphere. 11.8 ml of degassed DMF (bubbled with N for 30 minutes) was added to 2.554 g (5.88 mmol, 1 eq.) of 2, 2.240 g (8.82 mmol, 1.5 eq.) of bis(pinacolato)diboron, and 1.731 g (17.64 mmol, 3 eq.) of potassium acetate (dried under high vacuum). The mixture was stirred and bubbled with N for 10 minutes, after which 129 mg (0.18 mmol, 0.03 eq.) of Pd(dppf)Cl was added. The mixture was stirred and bubbled with N for an additional 10 minutes, after which the reaction was stirred in an oil bath at 90 °C for 10 hours in the absence of light. After cooling to room temperature, 25 mL of EtO was added, and the mixture was filtered through a thin layer of Celite, which was washed with 13 mL of EtO and 38 mL of cyclohexane. The filtrate was washed twice with 75 mL of H2O and once with 75 mL of brine, dried over MgSO4, and vacuum filtered through a pad of SiO2. The pad was washed with 75 mL of 1:1 Et2O / cyclohexane and evaporated under reduced pressure to ≤10 mL. The solution was stored overnight at 8 °C to give colorless white crystals, which were carefully broken up with a spatula and collected by vacuum filtration. After washing the crystals with two small portions of cold cyclohexane and air-drying to constant weight, 2.170 g (5.00 mmol, 85%) was obtained.

[0255]

number

[0256] (C directly bonded to boronic acid 5 arom (The signal is invisible) ESI-MS m / z (accurate mass): Calculated value: 246.13 [M-Boc-Gly] + , 335.15[M-Boc+H] + , 435.23[M+H] + , 457.21 [M+Na] + , 891.43 [2M+Na] + ;Actual values: 246.19, 334.94, 434.72, 456.95, 891.42.

[0257] Step 4: Synthesis of 4-bromo-2,6-dimethoxyaniline (4)

[0258] [ka]

[0259] 2.0 g (13.06 mmol, 1 equiv.) of 2,6-dimethoxyaniline was dissolved in 26 ml of acetic acid, and 3.52 ml (30.04 mmol, 2.3 equiv.) of 47% aqueous HBr was added with stirring. 3.02 g (16.98 mmol, 1.3 equiv.) of N-bromosuccinimide was added in one portion, and the reaction was stirred at room temperature for 1 hour. 125 ml of DCM was added, and the mixture was extracted three times with 125 ml of 1 M aqueous HCl. The combined aqueous extracts were cooled to 0°C, made strongly alkaline with stirring with 50% aqueous NaOH, and extracted three times with 50 ml of DCM. The combined organic extracts were dried over MgSO4 and filtered through a tightly packed pad of SiO2 in a sintered glass funnel, which was washed with an additional 100 ml of DCM. The filtrate was evaporated under reduced pressure to give 1.98 g (8.50 mmol, 65%) of a slightly brownish-reddish solid which initially appeared as an oil and crystallized spontaneously at room temperature.

[0260]

number

[0261] (The NH2 signal is not visible in CDCl3).

[0262]

number

[0263] ESI-MS m / z (accurate mass): Calculated value: 232.00[M( 79 Br)+H] + , 234.00 [M( 81 Br)+H] +, Actual values: 232.06, 233.96. Step 5: Synthesis of methyl 2-(4'-amino-4-(tert-butoxycarbonylamino)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetate (5)

[0264] [ka]

[0265] The following reaction steps were carried out under a N atmosphere. 1.950 g (4.49 mmol, 1 equiv.) of 3, 1.033 g (4.49 mmol, 1 equiv.) of 4, and 1.870 g of K2CO3 (13.47 mmol, 3 equiv.) were dissolved in 22.5 ml of degassed toluene (bubbled with N2 for 30 min) and 11.3 ml of HO (bubbled with N2 for 30 min). The mixture was stirred and bubbled with N2 for 10 min, after which 197 mg (0.27 mmol, 0.06 equiv.) of Pd(dppf)Cl2 was added. The biphasic mixture was stirred and bubbled with N2 for an additional 10 min, after which it was stirred in an oil bath at 90 °C for 5 h. After cooling to room temperature, the reaction was diluted with 200 ml of ethyl acetate and stirred until most of the solids dissolved. The biphasic mixture was vacuum filtered through a thin Celite pad and washed with 25 ml of 9:1 ethyl acetate / toluene and 125 ml of H2O. The filtrate was shaken and the phases separated. The organic layer was washed once with 125 ml of H2O and once with 125 ml of brine, dried over MgSO4, and filtered through a tightly packed pad of SiO2 in a sintered glass funnel, which was washed with an additional 100 ml of 9:1 ethyl acetate / toluene. The filtrate was evaporated under reduced pressure to give an oily brown solid (approximately 2 g). The solid was completely dissolved in refluxing isopropanol. The solution was evaporated under reduced pressure at 50 °C to approximately 10 ml (all in solution), heated to reflux with stirring, and then 50 ml of warm hexane was added with stirring. After stirring everything at reflux for 5 minutes, it was cooled to room temperature and kept at 4 °C overnight. The solid was collected by vacuum filtration, washed with two small portions of cold hexane, and air-dried to give 1.052 g (2.29 mmol, 51%) of a beige solid.

[0266]

number

[0267] (NH2 signal is invisible in CDCl3)

[0268]

number

[0269] ESI-MS m / z (accurate mass): Calculated value: 360.15[M-Boc+H] + , 460.21[M+H] + , 919.41[2M+H] + ;Actual values: 359.91, 460.01, 919.32.

[0270] Step 6a: Mn III Synthesis of Salophen Cl

[0271] [ka]

[0272] 2.163 g (20 mmol, 1 eq) of phenylenediamine and 4.175 ml (40 mmol, 2 eq) of salicylaldehyde were dissolved in 40 ml of EtOH and refluxed with stirring for 2 hours. The reaction mixture, containing an orange crystalline precipitate, was diluted with 460 ml of EtOH, 6.921 g (40 mmol, 2 eq) of Mn(OAc)2 was added, and the reaction mixture was refluxed with stirring for 5 hours under a slight air flow introduced through a condenser. 1.696 g (40 mmol, 2 eq) of LiCl was added, and stirring at reflux was continued for an additional 2 hours. The mixture was evaporated under reduced pressure to a volume of approximately 100 ml, and 500 ml of HO was added with stirring. The mixture was stirred vigorously at room temperature for 30 minutes. The precipitate was collected by vacuum filtration, washed three times with water, and air-dried to yield 6.050 g (14.95 mmol, 75%) of a light brown solid. The product was characterized solely by its catalytic activity according to Mirkhani et al., Bior Med Chem (2004), 12, 4376-7.

[0273] Step 6b: Synthesis of (E)-4'-(methyl 2-(4-(tert-butoxycarbonylamino)-3',5'-dimethoxybiphenyl-3-ylcarboxamide)acetate)-4'-(methyl 2-(4-(tert-butoxycarbonylamino)-3',5'-dimethoxybiphenyl-3-ylcarboxamide)acetate)diazene (6)

[0274] [ka]

[0275] Synthesis Procedure: Dissolve 19.3 ml of MeCN in 0.890 g (1.94 mmol, 1 equiv.) of 5, 39.2 mg (97.00 μmol, 0.05 equiv.) of Mn III To the solution was added salofen Cl and 6.6 mg (97.00 μmol, 0.05 equiv.) imidazole. After stirring the mixture for 5 minutes, 0.829 g (3.88 mmol, 2 equiv.) NaIO4 dissolved in 9.7 ml of HO was added, and the reaction was stirred at room temperature for 90 minutes. 80 ml of ethyl acetate was added, and stirring was continued for 5 minutes. The biphasic mixture was then filtered through Celite, which was washed twice with 10 ml of ethyl acetate. The filtrate was washed twice with 50 ml of HO and once with 50 ml of brine, dried over MgSO4, filtered, and evaporated under reduced pressure to yield the crude product as a dark solid. To a solution of the crude product in 5 ml of DCM, 50 ml of MeOH was added with stirring. The mixture was kept at -20 °C overnight, and the precipitate was collected by vacuum filtration and washed twice with a small amount of cold MeOH. After air-drying, 0.453 g (0.50 mmol, 51%) of a dark red solid was obtained. The mother liquor was evaporated to dryness under reduced pressure, and the residue was subjected to column chromatography (SiO, ethyl acetate / cyclohexane (2:1) + 1% AcOH). Evaporation of the solvent of the product-containing fractions under reduced pressure afforded an additional 0.275 g (0.30 mmol, 31%) of product as a wine-red solid and an overall yield of 0.728 g (0.80 mmol, 82%).

[0276]

number

[0277] ESI-MS m / z (accurate mass): Calculated value: 915.37[M+H] + , 1829.74[2M+H] + ;Actual values: 915.37, 1829.05. Step 7: Synthesis of (E)-4'-(2-(4-amino-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-amino-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene (7)

[0278] [ka]

[0279] 321 μl of acetyl chloride (4.5 mmol, 30 equiv.) was added to a solution of 182 μl (4.5 mmol, 30 equiv.) of dry MeOH in 1.15 ml of ethyl acetate at 0° C. The solution was left at 0° C. for 5 minutes and then added to 137.2 mg (0.15 mmol, 1 equiv.) of 6 dissolved in 1.5 ml of dry DCM at room temperature. The reaction was stirred at room temperature for 2.5 hours. The precipitated dianilinium chloride was collected by centrifugation, the supernatant was decanted, and the solid was washed twice with DCM by resuspension, centrifugation, and decantation. After air-drying and drying under reduced pressure, dianilinium chloride was obtained as a blue-purple solid in quantitative yield and used directly in the next step.

[0280] Therefore, the solid was dissolved / suspended in 1.5 ml of THF / MeOH (1:1) containing 150 μl (0.3 mmol, 2 equiv.) of 2 M aqueous NaOH solution. The mixture was stirred for 5 minutes, and 1.35 ml (2.7 mmol, 18 equiv.) of 2 M aqueous NaOH solution was added while stirring. The reaction was stirred at room temperature for 1 hour. Then, 1.5 ml of HO was added, and the reaction was stirred for an additional 1.5 hours. Then, 6 ml of HO was added, and the reaction was stirred for an additional 7 hours. After that, everything was dissolved by adding 7.5 ml of HO, the pH was adjusted to approximately 4 with 1 M aqueous HCl (compared to universal test paper), and the precipitate was collected by centrifugation. The supernatant was decanted, and the solid was washed three times with 5 ml of HO each by resuspension, centrifugation, and decantation. After drying under high vacuum, the product was obtained as a dark blue, highly static solid in quantitative yield (0.15 mmol, 103.0 mg).

[0281] The product was used directly in the next step without further purification or characterization. Step 8: Synthesis of (E)-4'-(benzhydryl 2-(4-amino-3',5'-dimethoxybiphenyl-3-ylcarboxamide)acetate)-4'-(benzhydryl 2-(4-amino-3',5'-dimethoxybiphenyl-3-ylcarboxamide)acetate)diazene (8)

[0282] [ka]

[0283] 103 mg (0.15 mmol, 1 equiv.) of 7 was dissolved in 750 μL of DMF. 750 μL of THF was added to the solution with stirring, followed by 450 μL (0.9 mmol, 6 equiv.) of 2 M diazodiphenylmethane in THF (Javed and Brewer, Org. Synth. (2008), 85, 189, DOI:10.15227 / orgsyn.085.0189). The reaction was stirred at room temperature for 15 hours, after which 225 μL (0.45 mmol, 3 equiv.) of 2 M diazodiphenylmethane in THF was added, and the reaction was stirred for an additional 7 hours. After this, 150 μl (0.3 mmol, 2 equiv.) of 2 M diazodiphenylmethane in THF was added, and the reaction was stirred for an additional 2 h. After this, 25 ml of cold EtO was added to precipitate the product. Precipitation was completed over 1 h at -20 °C, and the dark blue solid was collected by centrifugation. The supernatant was decanted, and the solid was washed twice with cold EtO by resuspension, centrifugation, and decantation. The solid was subjected to column chromatography (SiO, DCM / MeOH, step gradient of 0, 2, 4, …, 20% MeOH). The product-containing fractions were evaporated under reduced pressure, leaving 122 mg of a blue-reddish solid, consisting of the desired product and a significant amount of the product that had formed an imine with benzophenone (a trace of diimine was also present). The solid was dissolved in 750 μl of DMF, and 750 μl of THF was added to the solution with stirring, followed by 1.5 ml of 95% aqueous AcOH. After stirring the mixture at room temperature for 15 hours, 30 ml of cold EtO was added to precipitate the product. Precipitation was completed at -20°C for 1 hour, and the dark blue solid was collected by centrifugation. The supernatant was decanted, and the solid was washed twice with cold EtO by resuspension, centrifugation, and decantation. After removing residual solvent in vacuo, 107 mg (104.99 μmol, 70%) of the product was obtained as a dark blue solid, free of the imine by-product.

[0284]

number

[0285] ESI-MS m / z (accurate mass): Calculated value: 1019.39[M+H] + ;Actual value: 1019.68. Step 9: Synthesis of (E)-4'-(2-(4-(2-iodoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene (PS1)

[0286] [ka]

[0287] 102 mg (100.10 μmol, 1 equiv.) of 8 was dissolved in 2 ml of dry DCM, 34.7 mg (0.25 mmol, 2.5 equiv.) of KCO was added, and the mixture was stirred and cooled to 0° C. 18.4 μl (205.21 μmol, 2.05 equiv.) of iodoacetyl chloride was added, and the reaction was stirred at 0° C. for 1 h. An additional 1.34 μl (15.02 μmol, 0.15 equiv.) of iodoacetyl chloride was then added, and the mixture was stirred for an additional 1 h at 0° C., after which another 1.79 μl (20.03 μmol, 0.2 equiv.) of iodoacetyl chloride was added. After stirring at 0°C for an additional hour, the reaction was quenched by the addition of 1 ml of HO. The mixture was diluted with 18 ml of DCM and washed three times with 10 ml of HO by careful swirling and shaking to prevent emulsion formation. The organic phase was dried over MgSO and filtered through a thin layer of Celite, which was washed with DCM until the filtrate was colorless. Evaporation of the solvent under reduced pressure yielded a dark red-purple solid, which was dissolved in 1.5 ml of DCM with 0.1 ml of anisole. After cooling the solution to 0°C, 0.4 ml of TFA was added with mixing. The reaction was kept at 0°C for 2 hours, after which 20 ml of cold EtO was added to precipitate the product. Precipitation was complete over 1 hour at -20°C, and the dark purple solid was collected by centrifugation. The supernatant was decanted, and the solid was washed twice with cold EtO by resuspension, centrifugation, and decantation. After air drying and drying in vacuo, 102 mg (99.73 μmol, 99.6%) of a dark purple solid was obtained.

[0288]

number

[0289] ESI-MS m / z (accurate mass): Calculated value: 1023.06[M+H] + , Actual measurement: 1022.81 Example 2 Coupling with cysteine ​​side chains and photoinduced isomerization of 4'-(2-(4-(2-iodoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene (PS1) The UV-VIS absorption spectrum of azobenzene is π→π * and n → π * Two characteristic absorption bands corresponding to electronic transitions are revealed, which differ in amplitude and exact location of the absorption maximum (λ) for the trans and cis configurations. * is usually in the near-ultraviolet region around 340 nm (Sension et al., 1993), whereas the electronic transition n → π * is usually located in the visible (VIS) region around 420 nm and is due to the presence of a lone pair of electrons on the nitrogen atom (Nagele et al., 1997).

[0290] The key feature of the claimed photoswitchable azobenzene compounds designed to generate photoswitchable affinity matrices is the wavelength of light required to trigger photoisomerization and thus affinity modulation. Introducing electron-donating or push / pull substituents at the para position delocalizes the azobenzene chromophore, leading to longer-wavelength absorption, but also typically lowers the thermal barrier to isomer interconversion (Dong et al., 2015). Rapid thermal relaxation means that it is difficult to generate a large steady-state fraction of the cis isomer. Therefore, specifically preserving the binding activity of a photoswichtable affinity ligand with its cis isomer requires an impractically bright light source. Introducing substituents at all four ortho positions leads to azo compounds with several unusual properties useful for generating photoswitchable affinity matrices. Tetra-ortho-substituted azo compounds exhibit unusually slow thermal relaxation rates and enhanced n-π recombination of cis and trans isomers compared to analogs without ortho substituents. *The ortho-methoxy group greatly stabilizes the azonium form of the compound, where the azo group is protonated. Azonium ions absorb strongly in the red region of the spectrum and can reach the near-IR. These azonium ions can exhibit robust cis-trans isomerization in aqueous solution at neutral pH. By varying the nature of the ortho-substituents, along with the number and nature of the meta- and para-substituents, long-wavelength switching, stability against photobleaching, stability against hydrolysis, and stability against reduction by thiols can all be incorporated into the photoswitch (Dong et al., 2015).

[0291] HPLC and Spectroscopic Analysis: To test whether the synthesized PS1 was capable of responding to visible light-induced photoswitching when conjugated with a cysteine ​​side chain, the compound was coupled with N-acetylcysteine ​​(Fig. 3A), subjected to alternating irradiation cycles, and then analyzed.

[0292] In a typical experiment performed at 25°C, 1.0 mg of PS1 was dissolved in 97 μl of DMF in the absence of light to obtain a 10 mM solution. 0.5 μl of this solution was analyzed by HPLC using Method A (data not shown). 10 μl of this solution was combined with 100 μl of a 5 mM N-acetylcysteine ​​solution in aqueous buffer (100 mM Tris / Cl, 150 mM NaCl, 0.5% EDTA, pH 8.5). The mixture was shaken in the dark for 30 minutes. 5.5 μl aliquots of this solution were analyzed by HPLC using Method A (data not shown) and Method B. The remaining solution was illuminated with red light (LED-635 nm; NCSR219B-V1, Nichia Corporation, Tokushima, Japan) for 2 minutes, and 5.5 μl was analyzed by HPLC using Method B (Figure 3B). The solution was then illuminated with blue light (LED-465 nm; NCSR219B-V1, Nichia Corporation, Tokushima, Japan) for 2 min, and 5.5 μl was analyzed by HPLC using method B ( Figure 3B ).

[0293] The chromatogram in the top panel in Figure 3B reveals mostly trans isomers when dark-adapted, the chromatogram in the middle panel reveals mostly cis isomers with trans isomers as a minor species when illuminated at 635 nm, and the chromatogram in the bottom panel reveals mostly trans isomers with cis isomers as a minor species when illuminated at 465 nm.

[0294] At 25°C 1 / 2 To determine the cis → trans orientation, the solution was again illuminated with red light for 2 minutes, kept in the dark, and 5.5 μl aliquots were analyzed by HPLC method B at 0, 20, 40, ..., 120 minutes. 1 / 2 (cis → trans) was determined to be over 20 h at 25 °C (data not shown). UV-Vis spectra, HPLC-chromatograms and isomer ratios were recorded using a Thermo Scientific Dionex Ultimate 3000 HPLC system with a diode array detector.

[0295] Method A: Column: Agilent Zorbax Eclipse® XDB-C8 4.6 × 150 mm 5 micron, Eluent A: 10 mM NH4OAc in HO, Eluent B: 10 mM NH4OAc / MeCN (1:9) in HO, Gradient: 5 → 5% B (1 min) 5 → 95% B (12.5 min) 95 → 95% B (3.5 min), Flow rate: 2 ml / min.

[0296] Method B: Column: Agilent Zorbax Eclipse® XDB-C8 4.6 × 150 mm 5 micron, Eluent A: 10 mM NH4OAc in HO, Eluent B: 10 mM NH4OAc / MeCN (1:9) in HO, Gradient: 5 → 5% B (1 min), 5 → 20% B (12.5 min), 20 → 95% B (0.1 min), 95 → 95% B (3.4 min), Flow rate: 2 ml / min. The ratio of cis and trans isomers was determined by peak integration at 220 nm.

[0297] π-π around 340nm * The change in band intensity corresponds to photoswitching between the trans (high absorbance at 340 nm) and cis (low absorbance at 340 nm) conformations of PS1-AcCys in aqueous buffer (Figure 3C). High absorbance at 340 nm indicates the trans conformation, whereas low absorbance at 340 nm indicates the cis conformation.

[0298] Figure 3B shows the corresponding HPLC chromatogram with absorbance at λ = 220 nm (the wavelength at which trans-PS1-AcCys and cis-PS1-AcCys exhibit the same molar extinction coefficient, allowing direct comparison of peak integration). The chromatogram demonstrates that the cis- and trans-isomers of PS1-AcCys can be separated by HPLC (cis-PS1-AcCys t R = 9.1 min, trans-PS1-AcCys t R = 11.6 min). Irradiation with red light caused an increase in the proportion of cis-PS1-AcCys, here up to 88% (Figure 3B), which could be reversed by irradiation with blue light, thus restoring the ground state via photochemical reisomerization.

[0299] Thus, when the photoswitchable affinity matrix of the present invention is applied to affinity chromatography procedures, the cis configuration can correspond to a high-affinity state, whereas the trans configuration can correspond to a low-affinity conformation. High occupancy of each configuration was achievable by illumination with 635 nm and 465 nm light, respectively.

[0300] Example 3 Generation of SpA variant SpA#1 The Ig-binding protein of the present invention, as used in this example, comprises the B domain of Protein A (SEQ ID NO:4), defined as residues 215-268 corresponding to UniProtKB entry P38507 (SEQ ID NO:1). Residues 215-216 in SEQ ID NO:4 were replaced by Lys213-Ala214-Cys215-Gly216 (resulting in SEQ ID NO:6), and a single substitution, Ser250Cys, was further introduced to yield SpA#1 (SEQ ID NO:7). The sequence of the Ig-binding affinity ligand SpA#1 was extended at the N-terminus by Met212 and at the C-terminus by Ser269-Ala270-His271-His272-His273-His274-His275-His276.

[0301] To generate SpA variants suitable for derivatization with PS1, the DNA sequence encoding SEQ ID NO:4 was modified using a PCR assembly approach. First, two separate DNA fragments were generated, each sharing an overlapping sequence carrying the respective mutation. In a final PCR reaction, these two fragments were assembled to create a mutated DNA sequence that could serve as a PCR template for the introduction of further mutations or be subcloned into an expression vector.

[0302] To generate SpA#1, two consecutive mutations were introduced using the previously described approach. The wild-type DNA sequence was used as a PCR template, and two pairs of forward and reverse primers were used to introduce N-terminal protein sequence changes. The fragment was synthesized in a 20 μl PCR reaction containing 1× Phusion HF buffer, 0.5 μM forward and reverse primers, 5 ng of template DNA, 200 μM each dNTP, and 0.02 U of Phusion DNA polymerase per μl reaction mixture (New England Biolabs, Ipswich, Mass., USA). After an initial DNA denaturation at 98°C for 180 seconds, 30 PCR cycles were performed using a thermocycler, including steps of denaturation (10 seconds at 98°C), primer annealing (10 seconds at 61°C), and polymerization (15 seconds at 72°C). The reaction was completed after a final 180 seconds at 72°C. The fragments were assembled in a second 50 μl PCR reaction using 1 μl of each fragment reaction mixture and the flanking forward and reverse primers as template DNA. The reaction conditions were the same as before. All primers were supplied by MWG Eurofins (Ebersberg, Germany). The final PCR product was purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany).

[0303] The PCR fragment described above served as a template for the introduction of a second mutation (Ser250Cys) using a similar approach. A forward and reverse primer pair was used to generate the fragment. The flanking forward and reverse primers were used to assemble the final PCR product.

[0304] The mutated DNA fragment encoding SpA#1 was subcloned into a modified expression vector backbone based on pD451sr (ATUM, Newark, CA, USA) using the flanking DNA restriction sites XbaI and HindIII. The target vector and DNA insert were digested with the respective enzymes in a 50 μl reaction mixture (5 μg DNA; 1× CutSmart buffer; 0.04 U / μl XbaI; 0.04 U / μl HindIII-HF) and incubated at 37°C for 60 min. The digested DNA fragment was purified using a 1% (w / v) agarose gel and extracted using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The vector backbone and DNA insert were ligated in 20 μl of ligation reaction mixture (1× T4 DNA ligase reaction buffer; 100 ng of purified and linearized vector: 3 molar equivalents of purified DNA insert; 0.05 U / μl of T4 DNA ligase) and incubated at 20°C for 20 min. Transformation (Inoue et al., 1990) of chemically competent E. coli NEB Turbo cells (New England Biolabs, Ipswich, Mass., USA) was performed by adding 5 μl of the ligation reaction mixture to 50 μl of cell suspension, incubating on ice for 15 min, followed by heat shock at 42°C for 30 s and on ice for 30 s. After isolation of plasmid DNA from single clonal transformants, the correct sequence of the resulting expression plasmid pD451sr-SpA#1 was confirmed by Sanger sequencing.

[0305] The Cys-substituted residues were intended to provide a docking point for PS1 and preserve antibody binding when the photoswitch adopts the cis conformation (i.e., after illumination at 635 nm) but prevents binding in the trans conformation (i.e., after illumination at 465 nm) (Figure 5E). Cys215 is located at the N-terminus of the three-helix bundle, and Cys250 is located in the loop between helices 2 and 3. Isomerization of PS1 to the trans state should change the overall protein conformation and, therefore, the overall geometry required for interaction with the antibody molecule.

[0306] Example 4 Expression and purification of SpA#1 SpA variant SpA#1 was recombinantly produced as a soluble protein in the cytoplasm of Escherichia coli, isolated in high yield by immobilized metal ion affinity chromatography (IMAC) via a C-terminal 6xHis tag, purified by size-exclusion chromatography (SEC), and analyzed by SDS-PAGE.

[0307] First, a single colony of E. coli NEB T7 Express transformed with the expression plasmid pD451sr-SpA#1 encoding SpA#1 (SEQ ID NO: 7) was used to inoculate 3 ml of LB medium supplemented with 50 μg / ml kanamycin. After overnight incubation at 37°C and thorough agitation, 2 ml of the culture suspension was diluted into 200 ml of LB medium in a 1 L shake flask and again supplemented with 50 μg / ml kanamycin. The production culture was grown at 37°C and 250 rpm with orbital motion of 25 mm stroke until the optical density at 600 nm (OD600) reached 0.5. At this point, T7 promoter-controlled expression of SpA#1 was induced using 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and continued for an additional 4 hours without changing the growth conditions. At the end of the fermentation, E. coli cells were harvested by centrifugation (12,000 rcf, 10 min, 4°C). Sedimented cells were resuspended in 18 ml of cold IMAC buffer A (50 mM Tris / Cl pH 8.0 at 25°C; 150 mM NaCl; 20 mM imidazole) and lysed by adding 2 ml of 10x BugBuster (Merck Millipore). Further viscosity reduction was achieved with 0.4 units / ml of Benzonase (Merck Millipore). After incubation at 20°C for 20 min and gentle agitation, the crude extract was centrifuged (12,000 rcf, 20 min, 4°C) to sediment cell debris. The clarified supernatant was filtered using a PES membrane with a filtration precision of 0.22 μm.

[0308] Liquid handling during isolation of the target protein using IMAC was performed using an FPLC system (AKTA Pure 25, Cytiva Life Science) coupled with a 5 ml Ni-HisTrap HP column (Cytiva Life Science). The filtered supernatant containing the target protein SpA#1 was applied to an IMAC column pre-equilibrated with IMAC buffer A at a flow rate of 5 ml / min. Unbound protein was washed off with running buffer until a stable baseline was reached in the UV (280 nm) absorbance reading. Bound SpA#1 was step-eluted with elution buffer IMAC B, pH 8.0, containing 350 mM imidazole, 50 mM Tris / Cl, and 150 mM NaCl. Elution fractions containing SpA#1 were collected, supplemented with EDTA (5 mM final) and DTT (20 mM final), and analyzed by SDS-PAGE (Fling and Gregerson 1986). In a final purification step before derivatization with PS1, the IMAC eluate of SpA#1 was subjected to size-exclusion chromatography on a Superdex 75 Increase 10 / 300GL (Cytiva Life Science) column, which confirmed the monodispersity of the isolated SpA#1 and transferred the protein into coupling buffer (100 mM Tris / Cl pH 8.5; 150 mM NaCl; 0.5 mM EDTA).

[0309] Example 5 Derivatization of SpA#1 with PS1 A total volume of 3.8 ml of freshly degassed and reduced SpA#1 (304.2 μM, 1.16 μmol, 8.5 mg, 1 equiv.) was diluted with the same degassed buffer (100 mM Tris / Cl, 150 mM NaCl, 0.5 mM ETDA, pH 8.5) to a final protein concentration of 20 μM. The solution was stirred under a nitrogen atmosphere while 3.5 mg of PS1 (3.40 μmol, 3 equiv.) dissolved in 1.14 ml of DMF was added in 20 portions over 1 h. The reaction was kept in the dark and stirred at room temperature for an additional 2 h. We found that constant light irradiation was not necessary to maintain the cis conformation of PS1 during the reaction. The gradual addition of PS1 helped prevent the formation of doubly bridged species. The derivatization reaction yielded a large amount of monobridged species. Further purification by SEC was performed to remove trace amounts of cross-linked species and higher oligomers.

[0310] The precise chemical makeup of PS1-SpA#1 was analyzed by electrospray ionization mass spectrometry (ESI-MS) (Figure 4). ESI-MS analyses were performed using a Thermo Scientific LCQ-Fleet mass spectrometer coupled to a Thermo Scientific Dionex Ultimate 3000 HPLC system. These measurements revealed the correct covalent coupling of PS1 to SpA#1 (Figure 4B), accompanied by a mass increase of 767.26 Da.

[0311] Analysis data: ESI-MS m / z (accurate mass) of SpA#1: Calculated value: 671.71[M+11H] 11+ , 738.78[M+10H] 10+ , 820.75[M+9H] 9+ , 923.21[M+8H] 8+ , 1054.96[M+7H] 7+ , 1054.96[M+6H] 6+ ;Actual values: 671.33, 739.07, 821.07, 923.24, 1054.95, 1230.66. ESI-MS m / z (accurate mass) of PS1-SpA#1: Calculated value: 741.37[M+11H] 11+ , 815.40[M+10H] 10+ , 905.89[M+9H] 9+ , 1018.99[M+8H] 8+ , 1164.42[M+7H] 7+ , 1358.31[M+6H] 6+ ;Actual values: 741.68, 815.60, 906.58, 1019.68, 1165.13, 1358.81.

[0312] Example 6 Affinity purification of immunoglobulin G from cell culture supernatants using a photoswitchable affinity matrix Affinity resin preparation: The photoswitchable affinity ligand PS1-SpA#1 was immobilized on nickel-charged IMAC resin (Ni-Sepharose High Performance, Cytiva Life Sciences) via its C-terminal 6xHis tag. The resin was packed into a clear acrylic glass column with an internal diameter of 4 mm and a packed bed height of 20 mm, corresponding to a settled bed volume (SBV) of 250 μl. An LED array with a switchable peak wavelength of 635 nm (red) or 465 nm (blue) was mounted on the side of the column housing, surrounded by a reflective surface, to enable complete and sufficient illumination of the entire resin material within the column. The assembly was shielded from interfering stray light by the enclosure. First, the column was equilibrated with 20 CV of running buffer (50 mM Tris / Cl pH 8.5, 500 mM NaCl, 40 mM imidazole) at a constant flow rate of 1 ml / min, operated on an FPLC system (AKTA Pure 25, Cytiva Life Sciences). 1.2 mg of purified photoswitchable affinity ligand PS1-SpA#1 was then loaded onto the column. The flow-through was discarded and the column was washed with an additional 20 CV of running buffer.

[0313] Affinity purification of immunoglobulin G from cell culture supernatant: The chromatography was operated and monitored using an FPLC system (AKTA Pure 25, Cytiva Life Science). The absorbance at 280 nm was recorded to observe the course of protein adsorption and desorption (Figure 5A). A 10 ml sample of cell culture supernatant containing immunoglobulin G was dialyzed against the running buffer and loaded onto a photoswitchable affinity matrix (SpA#1-PS1) column at a flow rate of 0.5 ml / min (240 cm / h).

[0314] Unbound proteins and impurities (including host cell components) were washed off the column with 20 CV of running buffer. Sample application and washing steps were performed under visible (red) illumination at 635 nm.

[0315] Elution of bound immunoglobulin G was then induced by illumination of the resin with 465 nm visible light (blue). Such illumination altered the conformation of the binding protein in the affinity matrix in such a way that it lost its binding activity for the bound IgG, thus resulting in immediate elution (under constant buffer flow). Regeneration of the resin was achieved by illumination with 635 nm visible light (red). All chromatographic steps after loading were performed at a flow rate of 1 ml / min (480 cm / h) with the running buffer as the sole operating fluid. Elution fractions were collected and analyzed in terms of purity and yield using SDS-PAGE, SEC, and nanoDSF (Figure 5B, C, and D).

[0316] Sample analysis: SDS-PAGE analysis of the raw material and elution samples showed characteristic bands representing the heavy (approximately 50 kDa) and light (approximately 25 kDa) chains of the IgG molecule, indicating specific binding and subsequent light-controlled elution of immunoglobulin G. Furthermore, the sample collected under blue light (465 nm) illumination appeared to be of higher purity compared to the raw material (Figure 5B). The high protein quality of the isolated IgG was also demonstrated by analytical SEC of the elution sample (Figure 5C), which showed a monodisperse peak corresponding to the IgG molecule (approximately 150 kDa) without any detectable impurities, aggregates, or fragments. Additionally, the isolated IgG was analyzed by nanodifferential scanning fluorimetry (nanoDSF) using a Tycho NT.6 (NanoTemper Technologies, Munich, Germany). The recorded absorbance ratio (350 nm vs. 330 nm) indicated the immunoglobulin G molecule in its native folded state, along with the characteristic thermal denaturation events of the different antibody domains (FIG. 5D).

[0317] Taken together, the data collected during the isolation of human IgG1 using a light-controlled affinity matrix with the photoswitchable ligand PS1-SpA#1 demonstrate the general applicability of the present invention. Furthermore, the concept of a photoswitch that can bind to a protein ligand and modify its binding affinity for a target in a light-dependent manner appears plausible. The effect of affinity modification is most likely the result of distortion of the protein ligand secondary structure upon photoisomerization of the bound photoswitch PS1 (Figure 5E).

[0318] Example 7 Synthesis of 4'-(2-(4-(2-chloroacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene (PS2)

[0319] [ka]

[0320] 85.0 mg (123.80 μmol, 1 equiv.) of 4′-(2-(4-amino-3′,5′-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4′-(2-(4-amino-3′,5′-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene 7 was dissolved in 2.48 mL of dry DMF. The solution was stirred and cooled to 0° C. 24.6 μL (309.50 μmol, 2.5 equiv.) of chloroacetyl chloride was added and the reaction was stirred at 0° C. for 1 h. An additional 4.9 μL (0.5 equiv.) of chloroacetyl chloride was then added and the reaction was stirred for an additional 1 h at 0° C. before adding 9.8 μL (1 equiv.) of chloroacetyl chloride. After stirring for an additional hour at 0°C, another 4.9 μL (0.5 equiv.) of chloroacetyl chloride was added, and the reaction was stirred for an additional 7 hours at 0°C. 12.5 mL of cold 0.5 M aqueous NaH2PO4 (pH 3.5) was added, and stirring was continued for 5 minutes. The dark purple solid was collected by centrifugation and decantation of the supernatant, resuspended, washed three times with HO by centrifugation and decantation, and dried under vacuum at 50°C. The product was purified by dissolving the solid in 750 μL of DMF at 65°C, and 2.25 mL of warm CHCl3 was added, while stirring was continued for 5 minutes at 65°C. The mixture was cooled to room temperature and then held at 4°C for 15 hours. The dark blue solid was collected by filtration, washed twice with 400 μL of CHCl 3 , and dried at atmospheric pressure and then under high vacuum to yield 90.5 mg (108.78 μmol, 87%).

[0321] Analysis data:

[0322]

number

[0323] ESI-MS m / z (accurate mass): Calculated value: 839.18[M+H] + , Actual value: 839.27 Example 8 Generation of SpA variant SpA#2 The Ig-binding protein of the present invention comprises the B domain of protein A (SEQ ID NO: 4), defined as residues 215-268 corresponding to UniProtKB entry P38507. Residues 215-216 in SEQ ID NO: 4 were replaced by Lys213-Ala214-Cys215-Gly216. To provide a strategy for single-point covalent attachment via primary amines, all remaining lysine residues were substituted (Lys218Met, Lys246Arg, Lys260Gln, and Lys261Glu). A second cysteine ​​residue was introduced (Ser250Cys), resulting in SpA#2 (SEQ ID NO: 9). The sequence of the Ig-binding affinity ligand SpA#2 was extended at the N-terminus by Met212 and at the C-terminus by Ser269-Ala270-His271-His272-His273-His274-His275-His276.

[0324] To generate SpA#2 variants suitable for derivatization with PS1 and PS2, a DNA fragment encoding the SpA#1 variant was mutated via site-directed mutagenesis using appropriate mutagenic oligonucleotides, resulting in amino acid changes (Lys218Met, Lys246Arg, Lys260Gln, and Lys261Glu) relative to the sequence of SpA#1 (SEQ ID NO: 7) to generate SpA#2.

[0325] The mutated DNA fragment encoding SpA#2 was subcloned into a modified expression vector backbone based on pD451sr (ATUM, Newark, CA, USA) by seamless transfer via flanking SapI DNA restriction sites.

[0326] The Cys-substituted residues were intended to provide attachment points for PS1 or PS2 while preserving antibody binding when the photoswitch adopts a cis conformation (i.e., after illumination at 635 nm) but prevents binding in the trans conformation (i.e., after illumination at 465 nm). Position Cys215 is located at the N-terminus of the three-helix bundle, and position Cys250 is located in the loop between helices 2 and 3.

[0327] Example 9 Expression and purification of SpA#2 Following the experimental procedures described above, SpA variant SpA#2 was recombinantly produced as a soluble protein in the cytoplasm of E. coli, isolated in high yield by immobilized metal ion affinity chromatography (IMAC) via a C-terminal 6xHis tag, and analyzed by SDS-PAGE (analytical data not shown).

[0328] Example 10 Derivatization of SpA#2 with PS2 A total volume of 2 ml of freshly degassed and reduced SpA#2 (650 μM, 1.3 μmol, 10 mg, 1 equiv.) was diluted with the same degassed buffer (100 mM sodium borate, 150 mM NaCl, 1 mM TCEP, pH 8.5) to a final protein concentration of 50 μM. The solution was stirred under a nitrogen atmosphere while 2.2 mg of PS2 (2.6 μmol, 2 equiv.) dissolved in 0.26 ml of DMF was added in one portion. The reaction was illuminated with red light (635 nm) for 5 min and stirred at 35°C for 16 h. PS2 was found to be stable in the presence of 1 mM TCEP, which prevents the formation of disulfide-bridged species. The derivatization reaction results in a large amount of intramolecularly cross-linked species. Further purification steps by anion exchange chromatography and SEC were performed to remove minor cross-linked species and higher oligomers.

[0329] The precise chemical makeup of PS2-SpA#2 was analyzed by electrospray ionization mass spectrometry (ESI-MS). ESI-MS analysis was performed using a Thermo Scientific LCQ-Fleet mass spectrometer coupled to a Thermo Scientific Dionex Ultimate 3000 HPLC system. These measurements verified the successful covalent coupling of PS2 to SpA#2 by a mass increase of 767.26 Da (analytical data not shown).

[0330] Example 11 Affinity purification of immunoglobulin G from cell culture supernatant using PS2-SpA#2 Affinity resin preparation: The photoswitchable affinity ligand PS2-SpA#2 was covalently immobilized onto N-hydroxysuccinimide (NHS)-activated Sepharose (NHS-Sepharose Fast-Flow, Cytiva Life Sciences) via its N-terminus and its primary amine originating from Lys215.

[0331] The resin was packed into a clear acrylic glass column with an internal diameter of 4 mm and a packed bed height of 20 mm, corresponding to a settled bed volume (SBV) of 250 μl. An LED array with a switchable peak wavelength of 635 nm (red) or 465 nm (blue) was mounted on the side of the column housing, surrounded by a reflective surface, to enable complete and sufficient illumination of the entire resin material within the column. The assembly was shielded from interfering stray light by the enclosure. First, the column was equilibrated with 20 CV of running buffer (50 mM Tris / Cl pH 7.5, 150 mM NaCl) at a constant flow rate of 0.5 ml / min operated on an FPLC system (AKTA Pure 25, Cytiva Life Science). A 5 ml sample of cell culture supernatant containing immunoglobulin G was loaded onto the device at a flow rate of 0.5 ml / min. Unbound proteins and impurities were washed off the column with 20 CV of running buffer. Sample application and washing steps were performed under illumination with 635 nm visible light (red). Elution of bound immunoglobulin G was then triggered by illumination of the resin with 465 nm visible light (blue). Resin regeneration was performed by illumination with 635 nm visible light (red). All chromatographic steps after loading were performed at a flow rate of 0.5 ml / min with running buffer as the sole operating fluid. Elution fractions were collected and analyzed in terms of purity and yield using SDS-PAGE.

[0332] Sample analysis: SDS-PAGE analysis of the raw material and eluted samples showed characteristic bands representing the heavy (approximately 50 kDa) and light (approximately 25 kDa) chains of the IgG molecule, indicating specific binding and subsequent light-controlled elution of immunoglobulin G. The protein fraction eluted during illumination with blue light (465 nm) exhibited the highest protein purity, as demonstrated by SDS-PAGE analysis of the eluted fractions using PS2-SpA#2 as a photoswitchable affinity matrix (data not shown).

[0333] Additionally, the isolated IgG was analyzed by analytical SEC and nano-differential scanning fluorimetry (nano-DSF, data not shown) using a Tycho NT.6 (NanoTemper Technologies, Munich, Germany). The results showed that the protein quality was at the same level using PS2-SpA#2 instead of PS1-SpA#1 as the affinity matrix.

[0334] The type of bond formed between the solid support and the immobilized affinity ligand affects the performance of the photoswitchable affinity matrix in several ways. If the bond blocks or adversely affects the structure of the immobilized ligand, it will limit distortion of the protein ligand secondary structure upon photoisomerization of the attached photoswitch, thus allowing binding or elution of the target molecule. Bonds that allow the coupled ligand to leach from the matrix during operation or clean-in-place procedures will result in contamination of the purified protein and shorten the useful life of the affinity matrix.

[0335] These problems were addressed by the photoswitchable affinity ligands according to the third aspect of the present invention, which have none, only one, or a defined set of lysine residues. Covalent immobilization of the photoswitchable affinity ligand PS2-SpA#2 on a solid support via an amino-reactive linker (NHS) was possible with high precision. This further improvement of the photocontrolled affinity matrix paves the way for general application.

[0336] Example 12 4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene (PS3)

[0337] [ka]

[0338] 20.0 mg (29.13 μmol, 1 equiv.) of 4'-(2-(4-amino-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-amino-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene 7 was dissolved in 291 μL of dry DMF. The solution was cooled to -20 °C, and 22.7 mg (131.09 μmol, 4.5 equiv.) of 2,5-dihydro-2,5-dioxo-1H-pyrrole-1-acetyl chloride (Ilya Nifant'ev et al. (2021) Polymers, 13, 868, doi.org / 10.3390 / polym13060868) was added with mixing. The reaction was kept at -10 to -20 °C for 2 h. 1.45 mL of cold 0.5 M aqueous NaH2PO4 (pH 3.5) was added with mixing. The dark purple solid was collected by centrifugation and decantation of the supernatant, washed three times with HO by resuspension, centrifugation, and decantation, and dried under reduced pressure. Purification was carried out by column chromatography (SiO2, CHCl3 / MeOH containing 2.5% DMF and 2.5% AcOH, step gradient 0, 2, 4, 20% MeOH). Product-containing fractions were combined, and most of the CHCl3 and MeOH were removed under reduced pressure at 40 °C. The product was precipitated by the addition of cold Et2O (-20 °C), and precipitation was completed at -20 °C for 1 h. The solid was collected by centrifugation and decantation of the supernatant, washed twice with cold Et2O by resuspension, centrifugation, and decantation. The dark red solid was dried at atmospheric pressure and then under high vacuum to yield 15.4 mg (16.02 μmol, 55%).

[0339] Analysis data:

[0340]

number

[0341] ESI-MS m / z (accurate mass): Calculated value: 961.26[M+H] + , Actual value: 961.32 Example 13 Generation of a photoswitchable protein G variant (PS2-SpG#1) The immunoglobulin-binding protein described herein is composed of a C1 domain (SEQ ID NO: 12) identified in group C and group G streptococci. This domain is characterized by an immunoglobulin-binding region that exhibits specific affinity for both the Fc and Fab regions of antibodies. The affinity ligand detailed in this example encompasses a modified C1 domain lacking lysine residues, which have been replaced with alternative amino acids. This modification facilitates targeted covalent binding via primary amines. Additionally, substitution of Asn338 with Tyr (Asn338Tyr) improves stability against alkaline hydrolysis (SEQ ID NO: 15). To further this design, two cysteine ​​residues, Val322Cys and Asp348Cys, were introduced to generate the SpG#1 variant (SEQ ID NO: 18). The SpG#1 variant also features modifications at the N-terminus. The first Met301 and Ser302 residues were replaced with a short anchor peptide, MATKASK, followed by a polyglycine linker (GGGG), which provides a primary amine for solid support coupling. Additionally, the C-terminus was modified with up to eight negatively charged amino acids to aid in purification.

[0342] The SpG#1 variant was engineered for post-translational modification by PS2. Introduction of cysteine ​​residues at positions 322 and 348 creates anchoring points for PS2, which are intended to preserve antibody binding when the photoswitch is in the cis conformation (after illumination at 635 nm) and disrupt binding when the photoswitch is in the trans conformation (after illumination at 465 nm). The cysteine ​​residues are strategically positioned within the loop region interconnecting beta strand 2 with the alpha helix and between beta strands 3 and 4.

[0343] The protein variants were expressed as soluble fractions in the cytoplasm of Escherichia coli and achieved >90% purity via liquid chromatography. The purified protein was reduced using 20 mM dithiothreitol at a pH range of 7.5 to 8.5 for a minimum of 1 h at 25 °C. Excess reducing agent was then removed via buffer exchange using a HiPrep 26 / 10 desalting column (Cytiva Life Science). The derivatization reaction of the purified protein was carried out for 12 h under red light illumination. The reaction mixture consisted of 50 μM protein (SpG#1), a 2-fold molar excess of a photoswitchable azobiaryl compound (PS2) relative to 1 equivalent of protein, and a 10-fold molar excess of tris(2-carboxyethyl)phosphine hydrochloride (TCEP). This mixture was prepared in a degassed solution containing 10% (v / v) N,N-dimethylformamide (DMF), 100 mM NaCl, and 50 mM dimethylpiperazine (DMP), with the pH adjusted to 8.5 and the temperature set at 35 °C. Purification of the derivatization reaction mixture was performed through anion exchange chromatography using Capto Q ImpRes resin (Cytiva Life Science) to remove unreacted photoswitchable azobiaryl compound and unreacted protein, as well as minor cross-linked species and higher oligomers. The process utilized a gradient elution of 20 mM DMP buffer at pH 8.5, gradually increasing to 1 M NaCl. Fractions containing monomeric protein conjugated to a single molecule of PS2 were identified through SDS-PAGE and mass spectrometry analysis, then pooled and concentrated. ESI-MS analyses were performed using a Thermo Scientific LCQ-Fleet mass spectrometer coupled to a Thermo Scientific Dionex Ultimate 3000 HPLC system. These measurements verified the successful covalent coupling of PS2 to SpG#1 by a mass increase of 767.26 Da (analytical data not shown).

[0344] The derivatized and purified protein was then immobilized on NHS-agarose (NHS-Sepharose Fast-Flow, Cytiva Life Sciences) at pH 7.0 for a duration of at least 1 h, followed by quenching of excess reactive sites with 1 M ethanolamine at pH 9.5 for at least 1 h.

[0345] Example 14 Generation of a photoswitchable protein L variant (PS2-PpL#1) The immunoglobulin-binding proteins detailed herein include protein L, which was identified on the bacterial surface of Finegoldia magna. As characterized in Finegoldia magna strain 3316, protein L is notable for its four homologous immunoglobulin-binding domains, which exhibit specific affinity for immunoglobulin light chains. This specificity facilitates the purification of a broader range of antibody classes, such as IgA, IgM, IgE, and IgD, that are not amenable to binding by protein A or G. In addition, the light chain specificity of this Ig-binding affinity ligand allows for the purification of antibody fragments, including single-chain variable fragments (scFv) and Fab fragments, as well as fusions thereof. In this context, the affinity ligand incorporates the C2 domain of protein L, with its lysine residues changed to alternative amino acids other than lysine (SEQ ID NO: 17). This modification was strategically implemented to facilitate site-specific immobilization via amino-reactive chemistry, thereby expanding the usefulness of the ligand for light-controlled affinity purification processes.

[0346] Similar to the genetic engineering of SpG#1, the residue preceding Glu327 in the C2 domain of protein L was replaced with a short anchor peptide, MATKASK, followed by a flexible linker (GGGGAS), providing a primary amine for solid support coupling. Additionally, the C-terminus was modified with up to eight negatively charged amino acids to aid in purification. Furthermore, two cysteine ​​residues, Thr344Cys and Asp375Cys, were introduced to generate the PpL#1 variant (SEQ ID NO: 19).

[0347] The PpL#1 variant was specifically designed to undergo post-translational modification by the photoswitchable molecules PS1 and PS2. Strategic insertion of cysteine ​​residues at positions 344 and 375 serves as anchoring points for PS2. This design aims to maintain antibody binding affinity when the photoswitch is in its cis conformation, achieved after illumination at 635 nm, and disrupt this binding in the trans conformation after illumination at 465 nm. These cysteine ​​residues are strategically positioned in beta strands 2 and 3, which effectively span the outer strands of the beta sheet. The beta sheet then interacts with the alpha helix to form the characteristic fold of the C domain of protein L. This precise placement ensures effective modulation of binding properties through light-induced structural changes.

[0348] The protein was successfully expressed in E. coli following the previously described methodology, achieving a purity level of over 90%. After reduction with dithiothreitol (DTT) and subsequent buffer exchange, the protein underwent modification with PS2 as outlined in Example 13. Anion exchange chromatography utilizing Capto Q ImpRes resin (Cytiva Life Science) was used to purify the derivatization reaction mixture. This step was essential for the removal of unreacted photoswitchable azobiaryl compound, unreacted protein, and minor cross-linked species and higher oligomeric forms. The procedure employed 20 mM DMP buffer at a pH of 8.5, with a gradient elution gradually increasing to 1 M NaCl. Fractions containing monomeric protein conjugated to single molecules of PS2 were identified via SDS-PAGE and mass spectrometry, then pooled and concentrated for further analysis.

[0349] Electrospray ionization mass spectrometry (ESI-MS) analyses were performed using a Thermo Scientific LCQ-Fleet mass spectrometer in conjunction with a Thermo Scientific Dionex Ultimate 3000 HPLC system. These analyses confirmed successful covalent conjugation of PS2 to the PpL#1 variant, as evidenced by a mass increase of 767.26 Da (analytical data not shown).

[0350] After derivatization and purification, the protein was immobilized on NHS-agarose (NHS-Sepharose Fast-Flow, Cytiva Life Sciences) for a minimum of 1 h at pH 7.0. To ensure inactivation of any remaining reactive sites, a quenching step using 1 M ethanolamine at pH 9.5 was performed for at least 1 h to prepare the immobilized protein for subsequent applications.

[0351] Example 15 Affinity purification of immunoglobulin G from a complex polyclonal immunoglobulin mixture using PS2-SpG#1 and PS2-PpL#1 For functional evaluation of the photoswitchable affinity matrices, they were housed in a transparent acrylic glass enclosure featuring a thickness of 2 mm to achieve a settled bed volume (SBV) of 250 μl. An LED array equipped with a lens capable of alternating peak wavelengths of 635 nm (red) and 465 nm (blue) was positioned adjacent to the enclosure. This configuration ensured thorough and efficient illumination of the entire resin material contained therein. The performance of the photoswitchable affinity matrix was tested using the polyclonal antibody mixture Cutaquig® (Octapharma) as a substrate.

[0352] First, the column was equilibrated with 3 mL of running buffer (50 mM TrisHCl, pH 7.6, 150 mM NaCl) using an FPLC system (AKTA Pure 25, Cytiva Life Science). The affinity matrix was then loaded with the sample to achieve 60% breakthrough. Unbound proteins and impurities were removed by washing the column with 7.5 mL of the same running buffer. Both sample application and washing steps were performed under 635 nm red visible light illumination to ensure binding conditions. Following this, elution of bound immunoglobulin G (IgG) was initiated by switching the illumination to 465 nm blue visible light, thereby inducing the release of bound IgG. The resin was regenerated by re-illuminating with 635 nm red visible light, preparing it for subsequent use. Except for sample application, which was performed at 0.2 mL / min, all chromatographic process steps were performed at a constant flow rate of 0.5 mL / min, and running buffer was the only fluid used. Eluted fractions were collected for analysis of purity and yield via SDS-PAGE. The chromatographic profile obtained by this procedure is depicted in Figure 6.

[0353] The development of photoswitchable variants of proteins A, G, and L exemplifies the broad applicability of this invention. By using azobiaryl compounds (PS2) to modify proteins of various structures, we have successfully demonstrated a light-controlled affinity purification method. This work highlights the applicability of PS2 modifications for binding proteins to facilitate their light-regulated affinity modulation. Such a technique not only improves the efficiency of immunoglobulin G (IgG) purification but also paves the way for novel purification strategies utilizing light-responsive technologies, marking a significant advance in the field of biotechnology.

[0354] Example 16 4'-(2-(4-(2-chloroacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene (PS4)

[0355] [ka]

[0356] 100 mg (242.70 μmol, 1 equiv.) of 1-(4-bromo-2,6-difluorophenyl)-1-(4-bromo-2,6-difluorophenyl)diazene (S. Okumura et al., J. Org. Chem. 2013, 78, 12090–12105, doi:10.1021 / jo402120w), 221.3 mg (509.67 μmol, 2.1 equiv.) of 3, and 412.2 mg (1.94 mmol, 8 equiv.) of KPO were dissolved in 2.4 mL of degassed toluene and 1.2 mL of degassed HO. The mixture was stirred and bubbled with N for 10 min at room temperature. 17.8 mg (24.27 μmol, 0.1 equiv.) of Pd(dppf)Cl2 was added, and the mixture was stirred and bubbled with N2 for an additional 5 min. The reaction vessel was sealed under a N2 atmosphere and placed in an oil bath at 100 °C. The reaction was stirred for 2 h 30 min, after which it was cooled to room temperature. 20 mL of DCM and 5 mL of HO were added, and the pH of the aqueous layer was adjusted to 8–9 with 1 M aqueous HCl, followed by the addition of 2 mL of MeOH. The whole was filtered through a thin Celite pad, washed with 20 mL of HO, and the filter cake was extracted with two 25 mL portions of DCM / MeOH (10:1). The filtrate was shaken, the organic layer separated, dried over MgSO4, filtered, and the solvent evaporated under reduced pressure to afford 220 mg of crude product, which was purified by column chromatography (SiO2, DCM / ethyl acetate (12:1)). The solvent of the product-containing fractions was evaporated under reduced pressure to afford 104 mg of 9 (119.98 μmol, 49%) as a red solid containing the trans isomer and a small amount of the cis isomer.

[0357]

number

[0358] ESI-MS m / z (accurate mass): Calculated value: 867.30[M+H] + , 889.28[M+Na] + , Actual measurements: 866.92, 889.15

[0359] [ka]

[0360] 222 μL (3.11 mmol, 60 equiv.) of acetyl chloride was added to a solution of 126 μL (3.11 mmol, 60 equiv.) of dry MeOH in 0.79 mL of ethyl acetate at 0° C. The solution was left at 0° C. for 5 min and then added to 45 mg (51.94 μmol, 1 equiv.) of 9 suspended in 1.04 mL of dry DCM at room temperature. The reaction was stirred at room temperature for 13 h. The precipitated dianilinium chloride was collected by centrifugation, the supernatant was decanted, and the solid was washed twice with DCM by resuspension, centrifugation, and decantation. After air-drying and drying under reduced pressure, dianilinium chloride was obtained as a red solid in quantitative yield and used directly in the next step.

[0361] The solid was dissolved / suspended in 1.04 mL of THF / MeOH (1:1) containing 104 μL (207.76 μmol, 4 equiv.) of 2M aqueous NaOH. The mixture was stirred for 5 min. While stirring, 936 μL (1.87 mmol, 36 equiv.) of 2M aqueous NaOH was added, and the reaction was stirred at room temperature for 3 h. 9 mL of HO was added, and the reaction was stirred for an additional 30 min. The pH was adjusted to 3-4 with 1M aqueous HCl (compared to universal test paper), and the precipitate was collected by centrifugation. The supernatant was decanted, and the solid was washed three times with 5 mL of HO by resuspension, centrifugation, and decantation. After drying under vacuum at 40-45 °C, 10 was obtained as a red solid in quantitative yield (51.94 μmol) and used directly in the next step.

[0362] [ka]

[0363] 51.94 μmol (1 equiv.) of 10 was dissolved in 1.04 mL of dry DMF. The solution was stirred and cooled to 0°C. 20.7 μL (0.26 mmol, 5 equiv.) of chloroacetyl chloride was added, and the reaction was stirred at 0°C for 2.5 hours. 5 mL of 0.5 M aqueous NaH2PO4 (pH 3.5) was added, and stirring was continued for 10 minutes. The red solid was collected by centrifugation and decantation of the supernatant, washed three times with 5 mL of HO by resuspension, centrifugation, and decantation, and dried under reduced pressure. The product was purified by adding 450 μL of DMF to the solid and stirring at 65°C for 15 minutes. While stirring, 1.35 mL of warm CHCl3 was added, and stirring was continued at 65°C for 5 minutes. It was cooled to room temperature and then kept at 4°C for 6 hours. The red solid was collected by filtration, washed twice with 500 μL of CHCl 3 , and dried under a stream of air and then under high vacuum to yield 34 mg (42.96 μmol, 83%) of PS4 as a red solid.

[0364]

number

[0365] ESI-MS m / z (accurate mass): Calculated value: 789.09[MH] - , 394.04[M-2H] 2- , Actual values: 789.26, 394.38 Example 17 Photoinduced isomerization of 4'-(2-(4-(2-chloroacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene (PS4) A 1 mM solution of PS4 in DMF was prepared in the absence of light, and 5 μL of this solution was subjected to HPLC analysis as described in Example 2 with a 5 to 50% gradient of eluent B. The solution was then illuminated with yellow light (LED-593 nm, 350 mA), blue light (LED-450 nm, 350 mA), and UV light (312 nm, INTAS UV transilluminator) for 2 min each, and 5 μL of each illumination step was similarly subjected to HPLC analysis. Analysis showed good light-induced switching between the trans and cis states with the following trans / cis ratios: dark (92:8), yellow (14:86), blue (67:33), and UV (80:20).

[0366] At 25°C 1 / 2 To determine the cis → trans orientation, the solution was again illuminated with yellow light (LED-593 nm, 350 mA) for 2 min, kept in the dark, and 5 μL aliquots were analyzed by HPLC at 0, 20, 40, ..., 120 min. 1 / 2 (cis → trans) was determined to last for more than 1 day at 25°C.

[0367] The UV-Vis spectra of the cis and trans isomers were also recorded using a Thermo Scientific Dionex Ultimate 3000 HPLC system with a diode array detector (Figure 7).

Claims

1. Photoswitchable azobiaryl compounds of formula (I) 【Chemistry 1】 [In the formula, R ２ , ｎ , ２ , ３ , ｘ and R １ ’ are, independently, H, F, Cl, Br, (CH ２ ) ｎ CH ３ 、CH((CH ２ ) ｎ CH ３ )(CH ２ ) ｘ CH ３ ), C((CH ２ ) ｎ CH ３ )(CH ２ ) ｘ CH ３ )(CH ２ ) ｚ CH ３ ), (CH ２ ) ｎ CH((CH ２ ) ｘ CH ３ )(CH ２ ) ｚ CH ３ ), (CH ２ ) ｎ C((CH ２ ) ｘ CH ３ )(CH ２ ) ｙ CH ３ )(CH ２ ) ｚ CH ３ ), O(CH ２ ) ｎ CH ３ , OCH((CH ２ ) ｎ CH ３ )(CH ２ ) ｘ CH ３ ), OC((CH ２ ) ｎ CH ３ ) - ((CH ２ ) ｘ CH ３ )(CH ２ ) ｚ CH ３ ), O(CH ２ ) ｎ CH((CH ２ ) ｘ ) ｘ CH ３ (CH) ２ ) ｚ CH ３ )、O(CH ２ ) ｎ C((CH ２ ) ｘ CH ３ )-((CH ２ ) ｙ CH ３ (CH) ２ ) ｚ CH ３ )、S(CH ２ ) ｎ CH ３ 、SCH((CH ２ ) ｎ CH ３ (CH) ２ ) ｘ CH ３ )、SC((CH ２ ) ｎ CH ３ (CH) ２ ) ｘ CH ３ )-((CH ２ ) ｚ CH ３ )、S(CH ２ ) ｎ CH((CH ２ ) ｘ CH ３ (CH) ２ ) ｚ CH ３ )、S(CH ２ ) ｎ C((CH ２ ) ｘ CH ３ (CH) ２ ) ｙ CH ３ (CH) ２ ) ｚ CH ３ ) NH ２ ,NH(CH) ２ ) ｎ CH ３ 、NHCH((CH ２ ) ｎ CH ３ )-((CH ２ ) ｘ CH ３ )、NHC((CH ２ ) ｎ CH ３ (CH) ２ ) ｘ CH ３ (CH) ２ ) ｚ CH ３ )、NH(CH ２ ) ｎ CH((CH ２ ) ｘ CH ３ (CH) ２ ) ｚ CH ３ )、NH(CH ２ ) ｎ C-((CH) ２ ) ｘ CH ３ )-((CH ２ ) ｙ CH ３ (CH) ２ ) ｚ CH ３ )、N((CH ２ ) ｎ CH ３ ) ２ N(CH(CH ２ ) ｎ CH ３ (CH) ２ ) ｘ CH ３ )) ２ N(C(CH) ２ ) ｎ CH ３ )-((CH ２ ) ｘ CH ３ (CH) ２ ) ｚ CH ３ )) ２ N(CH) ２ ) ｎ CH((CH ２ ) ｘ CH ３ (CH) ２ ) ｚ CH ３ )) ２ N(CH) ２ ) ｎ C((CH ２ ) ｘ CH ３ ) ((CH ２ ) ｙ CH ３ )-((CH ２ ) ｚ CH ３ )) ２ , aziridin-1-yl, azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, azepan-1-yl, azocan-1-yl, morpholin-1-yl, 4-methyl-piperazin-1-yl; where n can be any integer from 0 to 10, x may be any integer from 0 to 10; y can be any integer from 0 to 10; z can be any integer from 0 to 10; R １ or R １’ At least one of them is not H, R ２ and R ２’ are independently SO ３ H, SO ３ Li, S.O. ３ Na, SO ３ K, COOH, COONHS, CONH ２ , CONH-(CH ２ ) ｎ CH ３ ,CONH-((CH ２ ) ｎ CH ３ ) ((CH ２ ) ｚ CH ３ ), CONH-(CH ２ ) ｎ SO ３ H,CONH-(CH ２ ) ｎ SO ３ Li, CONH-(CH ２ ) ｎ SO ３ Na,CONH-(CH ２ ) ｎ SO ３ K,CONH-(CH ２ ) ｎ CCH, CONH-(CH ２ ) ｎ N ３ , CONH-(CH ２ ) ｎ NH ２ , CONH-(CH ２ ) ｎ COOH, CONH-(CH ２ ) ｎ NHAcI, CONH-(CH ２ ) ｎ NHAcBr, CONH-(CH ２ ) ｎ NHAcCl, CONH-(CH ２ ) ｎ N(maleimide), CONH-(CH ２ ) ｎ —NH(2-chloromethyl acrylate), CONH—(CH ２ ) ｎ -NH(vinyl sulfonate), CONH-(CH ２ ) ｎ -NHCOPhF ５ 、CONH-(CH ２ ) ｎ -NHSO ２ PhF ５ 、CONH-(CH ２ ) ｎ COONHS、CONH-PEG-OH、CONH-PEG-NH ２ 、CONH-PEG-COOH、CONH-PEG-COONHS、CONH-PEG-O(CH ２ ) ｎ So ３ H, CONH-PEG-O (CH) ２ ) ｎ So ３ Li, CONH-PEG-O (CH) ２ ) ｎ So ３ Na, CONH-PEG-O (CH) ２ ) ｎ So ３ K, CONH-PEG-NHACI, CONH-PEG-NHACBr, CONH-PEG-NHACCl, CONH-PEG-N (Majimda), CONH-PEG-NH(2-クロロメチルアクリレート)、CONH- PEG-NH(ビニルスルホネート)、CONH-PEG-NHCOPhF ５ 、CONH-PEG-NHSO ２ PhF ５ 、CONH-PEG-N ３ 、CONH-PEG-OCH ２ CCH、CONH-PEG-NHCH ２ CCH、CONH-PEG-N(CH ２ CCH ２ 、CONH-PEG-NHCO(CH ２ ) ｎ COOH、CONH-PEG-NHCO(CH ２ ) ｎ COONHS、CONH-(Xaa) ｎ - OH, CONH - (Yaa) ｎ - NH (CH) ２ ) ｎ So ３ H、CONH-(Xaa) ｎ - NH (CH) ２ ) ｎ So ３ Li, CONH-(Xaa) ｎ -NH(CH ２ ) ｎ SO ３ Na, CONH-(Xaa) ｎ -NH(CH ２ ) ｎ SO ３ K, CONH-(Xaa) ｎ -OMe, CONH-(Xaa) ｎ -ONHS, CONH-(Xaa) ｎ -ONHS, CONH-(Xaa) ｎ -NH(CH ２ ) ｎ CCH, CONH-(Xaa) ｎ -NH(CH ２ ) ｎ N ３ , CONH-(Xaa) ｎ -NH(CH ２ ) ｚ NHAcI, CONH-(Xaa) ｎ -NH(CH ２ ) ｚ NHAcBr, CONH-(Xaa) ｎ -NH(CH ２ ) ｚ NHAcCl, CONH-(Xaa) ｎ -NH(CH ２ ) ｚ -N(maleimide), CONH-(Xaa) ｎ -NH(CH ２ ) ｚ NH(2-chloromethyl acrylate), CONH-(Xaa) ｎ -NH(CH ２ ) ｚ NH(vinyl sulfonate), CONH-(Xaa) ｎ -NH(CH ２ ) ｚ NHCOPhF ５ , CONH-(Xaa) ｎ -NH(CH ２ ) ｚ NHSO ２ PhF ５ , CONH-(Xaa) ｎ -N ３ , CONH-(Xaa) ｎ C ２ [#|、||||) ｎ !H ２ [#|、||||) ｎ (CH ２ pi?) ２ 、; ｎ H-(H ２ ) ｎ The ｎ H-(H ２ ) ｎ The ｎ ____________________________) ｎ _____________________________________ ２ 、; ｎ _____________________________) ｎ ___________________________) ｎ ____________________________) ｎ ____________________) ｎ __________________________) ｎ -NH-PEG-N(マレイミド)、CONH-(Xaa) ｎ ____________________________________________ ｎ _______________________________________________________ ｎ ___________________________________ ５ 、; ｎ _______________________________ ２ (F ５ 、; ｎ ________________________________ ３ 、; ｎ ___________________________________ ２ [#|、||||) ｎ _____________________________________ ２ [#|、||||) ｎ !PPPPH ２ pi?) ２ ,CONH-(Xaa) ｎ -NH-PEG-NHCO(CH ２ ) ｎ COOH,CONH-(Xaa) ｎ -NH-PEG-NHCO(CH ２ ) ｎ COONHS, where Xaa can be any standard or non-standard amino acid; n can be any integer from 0 to 10; z can be any integer from 0 to 10; R ３ and R ３’ are independently H, NH ２ , NHCO(C １ ~C ６ -alkyl), NHCO(C １ ~C ６ -haloalkyl), NHBoc, NHCbz, NHalloc, NH(CH ２ ) ｎ CH ３ , NHCH((CH ２ ) ｎ CH ３ ) ((CH ２ ) ｘ CH ３ ), NHC ((CH ２ ) ｎ CH ３ ) ((CH ２ ) ｘ CH ３ ) ((CH ２ ) ｚ CH ３ ), NH(CH ２ ) ｎ CH ((CH ２ ) ｘ CH ３ ) ((CH ２ ) ｚ CH ３ ), NH(CH ２ ) ｎ C((CH ２ ) ｘ CH ３ ) ((CH ２ ) ｙ CH ３ ) ((CH ２ ) ｚ CH ３ ), N((CH ２ ) ｎ CH ３ ) ２ , N(CH((CH ２ ) ｎ CH ３ )-((CH ２ ) ｘ CH ３ )) ２ , N(C((CH ２ ) ｎ CH ３ ) ((CH ２ ) ｘ CH ３ ) ((CH ２ ) ｚ CH ３ )) ２ , N((CH ２ ) ｎ CH ((CH ２ ) ｘ CH ３ )-((CH ２ ) ｚ CH ３ )) ２ , N((CH ２ ) ｎ C((CH ２ ) ｘ CH ３ ) ((CH ２ ) ｙ CH ３ ) ((CH ２ ) ｚ CH ３ )) ２ , aziridin-1-yl, azetidin-1-yl, pyrrolidin-1-yl, piperidin-1-yl, azepan-1-yl, azocan-1-yl, morpholin-1-yl, 4-methyl-piperazin-1-yl, NHCH ２ CHCH ２ , N(CH ２ CHCH ２ ) ２ , NHCH ２ CCH, N(CH ２ CCH) ２ , NHCOCCH, NHCO(CH ２ ) ｎ N ３ , NH acrylate, NH (2-chloromethyl acrylate), NH (vinyl sulfonate), N (maleimide), N (2-bromomaleimide), N (2,3-dibromomaleimide), NHCO aryl, NHCO haloaryl, NHSO ２ Aryl, NHSO ２ Haloaryl, (CH ２ ) ｎ N(maleimide), (CH ２ ) ｎ N(2-bromomaleimide), (CH ２ ) ｎ N(2,3-dibromomaleimide), NHCO(CH ２ ) ｎ N(maleimide), NHCO(CH ２ ) ｎ N(2-bromomaleimide), NHCO(CH ２ ) ｎ N(2,3-dibromomaleimide), NCS, NCO, NH(CH ２ ) ｎ CH(O)CH ２ , N((CH ２ ) ｎ CH(O)CH ２ ) ２ , NAcCH ２ CH(O)CH ２ and selected from the group comprising: where n may be any integer from 0 to 10, and z may be any integer from 0 to 10.

2. Aryl １ -aryl ２ and aryl １’ -aryl ２’ is a polyfunctionalized biaryl moiety, preferably wherein at least one R １ and at least one R １’ is not hydrogen, and more preferably both R １ and both R １’ is not hydrogen, and / or R ２ and R ２’ at least one, preferably both, of is CONH-Xaa-OH (Xaa = any standard or non-standard amino acid) or R ２ and R ２’ at least one, and preferably both, of the above comprises a linker selected from the group consisting of a peptide, a bifunctional alkane, and a poly(alkylene oxide), preferably wherein said poly(alkylene oxide) has a molecular weight selected from the group consisting of about 100 g / mol to about 80,000 g / mol, more preferably about 100 g / mol to 6,000 g / mol; and / or R ３ and R ３’ At least one, preferably both, of the amines according to claim 1, acrylamide, NHCO(C １ ~C ６ -haloalkyl), vinylsulfonate, isothiocyanate, isocyanate, epoxide, maleimide, haloarylcarboxy- and haloarylsulfonamide; The photoswitchable azobiaryl compound of claim 1.

3. 3. The photoswitchable azobiaryl compound of claim 1, which is 4'-(2-(4-(2-iodoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene.

4. 3. The photoswitchable azobiaryl compound of claim 1, which is 4'-(2-(4-(2-chloroacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene.

5. 3. The photoswitchable azobiaryl compound of claim 1, which is 4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dimethoxybiphenyl-3-ylcarboxamido)acetic acid)diazene.

6. The compound is 4'-(2-(4-(2-iodoacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene; or the compound is 4'-(2-(4-(2-chloroacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene; or the compound is 4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-difluorobiphenyl-3-ylcarboxamido)acetic acid)diazene; or the compound is 4'-(2-(4-(2-iodoacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-iodoacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)diazene; or the compound is 4'-(2-(4-(2-chloroacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2-chloroacetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)diazene; or the compound is 4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)-4'-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-3',5'-dichlorobiphenyl-3-ylcarboxamido)acetic acid)diazene; 3. A photoswitchable azobiaryl compound according to claim 1 or 2.

7. A photoswitchable affinity ligand comprising an affinity ligand stably associated with a photoswitchable azobiaryl compound according to any one of claims 1 to 6.

8. 8. The photoswitchable affinity ligand of claim 7, wherein the photoswitchable azobiaryl compound is stably associated with two conjugation sites in the affinity ligand in a bifunctional or bifunctional manner.

9. 9. The photoswitchable affinity ligand of claim 7, wherein the affinity ligand is selected from the group comprising immunoglobulin (Ig) binding proteins, preferably selected from the group comprising protein A, protein G and protein L, or variants thereof capable of specifically binding to immunoglobulins.

10. The affinity ligand comprises the B domain of protein A (SEQ ID NO: 4), which may be substituted with up to two cysteine ​​residues, or a protein domain having at least 80% sequence identity with SEQ ID NO: 4; preferably, the affinity ligand comprises a protein domain having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7; more preferably, the affinity ligand comprises a protein domain having the amino acid sequence of SEQ ID NO: 7; Alternatively, the affinity ligand comprises the lysine-poor B domain of protein A (SEQ ID NO: 5), optionally substituted with up to two cysteine ​​residues, or comprises a protein domain having at least 80% sequence identity with SEQ ID NO: 5; preferably, the affinity ligand comprises a protein domain having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11; more preferably, the affinity ligand comprises a protein domain having the amino acid sequence of SEQ ID NO: 11; Alternatively, the affinity ligand comprises at least one of the three homologous domains of protein G defined as C1 (SEQ ID NO: 12), C2 (SEQ ID NO: 13) and C3 (SEQ ID NO: 14), which may have up to two substitutions of the wild-type residues by cysteines, or comprises a protein domain having at least 80% sequence identity with SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14, or the affinity ligand comprises the lysine-poor domain of protein G defined as C1 (SEQ ID NO: 15), which may have up to two substitutions of the wild-type residues by cysteines, or comprises a protein domain having at least 80% sequence identity with SEQ ID NO:

15. Alternatively, the affinity ligand comprises the C domain of protein L or a protein domain having at least 80% sequence identity thereto, which may have two substitutions of wild-type residues by up to two cysteines, preferably the C domain of protein L is C2 (SEQ ID NO: 16) or a protein domain having at least 80% sequence identity thereto. Alternatively, the affinity ligand comprises a lysine-poor C domain of protein L, which may have two substitutions of wild-type residues by up to two cysteines, or a protein domain having at least 80% sequence identity therewith; preferably, the C domain of protein L is C2 (SEQ ID NO: 17) or a protein domain having at least 80% sequence identity therewith. A photoswitchable affinity ligand according to any one of claims 7 to 9.

11. 1) a solid support; ２） a) a photoswitchable azobiaryl compound according to any one of claims 1 to 6 in stable association with an affinity ligand, wherein the photoswitchable azobiaryl compound and / or the affinity ligand is in stable association with the solid support; or b) a photoswitchable affinity ligand according to any one of claims 7 to 10 stably associated with said solid support; and A photoswitchable affinity matrix comprising:

12. Use of a photoswitchable compound to isolate and / or purify a target molecule.

13. 13. Use of a photoswitchable compound according to claim 12, wherein the photoswitchable compound is a photoswitchable azobiaryl compound comprising at least two substituted biphenyl moieties, preferably the photoswitchable compound is a photoswitchable azobiaryl compound according to any one of claims 1 to 6.

14. Use of a photoswitchable affinity ligand, comprising an affinity ligand in stable association with a photoswitchable compound, to isolate and / or purify a target molecule.

15. Use of a photoswitchable affinity ligand as described in claim 14, wherein the photoswitchable affinity ligand comprises an affinity ligand stably associated with a photoswitchable azobiaryl compound comprising at least two substituted biphenyl moieties, preferably the photoswitchable affinity ligand is a photoswitchable affinity ligand as described in any one of claims 7 to 10.

16. Use of a photoswitchable affinity matrix according to claim 11 for isolating and / or purifying a target molecule.

17. 1. A method for isolating and / or purifying a target molecule, comprising: a) providing a composition comprising a target molecule; b) contacting the composition with an affinity matrix for a time sufficient to allow specific binding of the target molecule to the affinity matrix, the affinity matrix comprising a photoswitchable compound stably associated with an affinity ligand and a solid support to form an affinity matrix; c) washing the affinity matrix with a wash solution to remove components of the composition that are not specifically bound to the affinity matrix; d) irradiating the affinity matrix with light of a wavelength of at least about 400 nm to cause the affinity matrix to lose its specific binding to the target molecule; e) eluting the target molecule from the affinity matrix using an eluent; A method comprising:

18. 1. A method for isolating and / or purifying a target molecule, comprising: a) providing a composition comprising a target molecule; b) contacting the composition with an affinity matrix for a time sufficient to allow specific binding of the target molecule to the affinity matrix, wherein the affinity matrix comprises: i) a photoswitchable azobiaryl compound comprising at least two substituted biphenyl moieties, preferably a photoswitchable azobiaryl compound according to any one of claims 1 to 6 in stable association with an affinity ligand, wherein said photoswitchable azobiaryl compound and said affinity ligand are in stable association with a solid support to form an affinity matrix; or ii) a photoswitchable affinity ligand according to any one of claims 7 to 10, which is stably associated with a solid support to form an affinity matrix; or iii) A photoswitchable affinity matrix according to claim 11. and c) washing the affinity matrix with a wash solution to remove components of the composition that are not specifically bound to the affinity matrix; d) irradiating the affinity matrix with light of a specific wavelength to cause the affinity matrix to lose its specific binding to the target molecule; e) eluting the target molecule from the affinity matrix using an eluent; A method comprising:

Images