Polypeptides having NH-triazole functionalizations

Abstract

The present invention provides polypeptides comprising a NH-1,2,3-triazole, two alternative methods for synthesizing such polypeptides and a method for preparing a polypeptide being a stabilized variant of a protein having a certain function, which method comprises introducing at least one 1H-1,2,3-triazole moiety into said protein, thereby providing a polypeptide having increased stability and substantially the same biological function. Furthermore, the invention provides a metal affinity chromatographic method for purifying a polypeptide being NH-1,2,3-triazole functionalized.

Description

[0001] P481445PC00

[0002] Polypeptides having NH-triazole functionalizations.

[0003] Technical field of the invention

[0004] The present invention relates to the the field of protein chemistry, organic chemistry, biotechnology and pharmaceuticals. In particular the present invetion relates to polypeptides being modified to comprise non-natural functionalization such as to improve their properties.

[0005] Background

[0006] Recent advances in designing and producing proteins have led to this class of biomolecules going beyond their natural occurrence, even further, by incorporating non-natural fragments into their structure.1" Biosynthetic and, more recently, chemical methods1" for the synthesis of proteins and the post-translational modification of native forms1'' '' are tools for incorporation of these fragments into proteins with the aim of improving their structural properties, e.g. their thermal and chemical stability, as well as their function, e.g. catalytic efficiency, binding to target receptors, or even introducing new functionalities. Therefore, these strategies open avenues for novel protein functions, with some fragments having high functional potential even when present in small amounts in the protein structure.

[0007] Histidine, for example, has a variety of roles in proteins and is considered one of the key residues for their function and structure resulting from its specific side chain imidazole (Fig. la). ''1''" Imidazole is a 5-membered aromatic heterocycle, with two non-adjacent nitrogen atoms in metaposition, with a pKb of 7.0 for its conjugate acid and pKa of 14.5, regulating several pH dependent functions. Imidazole binds metal ions and serves as both an acceptor and donor of hydrogen bonds. It also forms cation-n bonds with metal ions or positively charged amino acids and n-n stacking interactions with aromatic side chains. The imidazole is a key residue in many different enzymatic reactions, as the lone pair of the sp2-hybridized nitrogen atom serves as a base in the catalytic pockets of proteases, as a key residue for long-range proton transfer, and as a ligand for metal ions in metalloenzymes. In addition, the ability of imidazole to coordinate metal ions is being explored to improve the mechanical properties of living organisms, such as high hardness and extensibility, inspiring advanced supramolecular materials, and is also being explored for purification in laboratory protein expression through His-tag.v P481445PC00

[0008] Its close structural non-natural analog NH-triazole (lH-l,2,3-triazole) has similar yet slightly different structural properties due to an additional pyridine-like nitrogen atom (Fig. la).xCompared to imidazole, NH-triazole is more acidic, with a pKa of 9.4 and pKb of 1.2 for its conjugate acid. Similar to imidazole, it binds strongly to metal ions and it has an additional potential binding site due to the additional nitrogen atom, so that it can bind two metals simultaneously.XI,X" As it is more acidic compared to imidazole, it can bind metals in a much wider pH range. NH-triazole also forms hydrogen bonds and participates in n-stacking due to its aromatic character. However, the chemistry of NH-triazole in the context of biomolecules is completely unexplored due to the lack of synthetic methods for its incorporation into more complex structures, as its incorporation into moderately functionalized small molecules is already challenging / 1"

[0009] Proteins and polypeptides are widely used as pharmaceuticals where a key challenge remains their stability both with respect to the drug substance and the drug product (during manufacture and during shelf-life of the product) as well as upon administration of the product. Hence, there is a need in the art to provide polypeptides having increased stability.

[0010] Summary of the invention

[0011] An object of the present invention is to provide polypeptides having increased stability. It is also an object of the invention to provide such stabilized polypeptides which substantially retain their original biological activity.

[0012] The present inventors have surprisingly found that incorporating NH-triazole into proteins renders these modified polypeptides much more stable. Herein, we report two synthetic strategies for the incorporation of NH-triazole, an imidazole analogue, into proteins. Site-specific incorporation can be achieved by a copper-catalyzed reaction of in situ formed hydrogen azide (hydrazoic acid, HN3) with an alkyne-functionalized protein (Fig. lb), while direct conjugation to native protein amine sites is achieved with an NH-triazole-functionalized NHS esters (Fig. lc). Using six structurally diverse protein substrates, we show that these synthetic strategies are efficient (>70% yields) and do not perturb native protein structure due to the mild reaction conditions occurring in an aqueous medium. Finally, we show that the incorporation of three NH-triazoles into human insulin resulted in superior thermal and chemical stability, while retaining its structure and biological activity even after heating to 95 °C (Fig. Id). The presented strategies provide a simple way to introduce nonnatural NH-triazole group into proteins to improve their chemical and functional properties. P481445PC00

[0013] In a first aspect the present invention provides a polypeptide comprising a NH-l,2,3-triazole.

[0014] In one embodiment the polypeptide is a derivative of human insulin, desB30-human insulin, or a human insulin analogue.

[0015] In a second aspect the present invention provides a method for preparing the polypeptide as defined in the first aspect, comprising the steps : a) providing the protein to be NH-l,2,3-triazole functionalized, b) introducing at least one alkyne into the protein to form an alkyne-functionalized protein, c) reacting the alkyne functionalized protein with hydrogen azide in the presence of a copper catalyst, and c) isolating said polypeptide.

[0016] In a third aspect the present invention provides a method for preparing a polypeptide as defined in the first aspect, comprising the steps : a) providing the protein to be NH-l,2,3-triazole functionalized, b) providing a NH-l,2,3-triazole functionalized NHS ester, c) reacting the protein with NH-l,2,3-triazole functionalized NHS ester, and d) isolating said polypeptide.

[0017] In a fourth aspect the present invention provides a method for preparing a polypeptide being a stabilized variant of a protein having a certain biological function, which method comprises introducing at least one lH-l,2,3-triazole moiety into said protein, thereby providing a polypeptide having increased stability and substantially the same biological function.

[0018] In a fifth aspect the present invention provides a method for purifying a polypeptide which comprises at least one lH-l,2,3-triazole moiety, wherein said method comprises metal affinity chromatography.

[0019] In a sixths aspect the present invention provides a pharmaceutical formulation comprising the polypeptide as defined in the first aspect, and at least one pharmaceutically acceptable excipient.

[0020] In some embodiments the pharmaceutical formulation is an aqueous formulation. P481445PC00

[0021] Figure 1. a, Structural features of NH-triazole and imidazole. Two strategies for incorporating NH- triazole into proteins, b, by copper-catalyzed reaction of hydrogen azide (HN3) formed in situ from sodium azide (NaN3) with a protein with pre-installed alkyne handle, or c, by reaction of NH-triazole functionalized NHS ester with basic residues of the protein, d, Introduction of A / H-triazoles with appropriate linker lengths at the N-terminus of the A and B chains, i.e. Al-Gly, Bl-Phe, and B29-Lys results in thermally stable insulin, which exhibits biological activity even after exposure to high temperatures.

[0022] Figure 2. a, General reaction scheme for the copper-catalyzed cycloaddition of hydrogen azide to the alkyne of the protein, b, The reaction depends on 4 key parameters, i.e. (i) pH 5 of the buffer medium providing sufficient HN3concentration, (ii) Na(asc) for Cu(l) formation and as scavenger of ROS, (iii) and Cu ions in combination with THPTA, all of which are required for (iv) successful reaction. [Cu, HN3] indicates reaction conditions described in Fig. 2a. c, Reaction with RimM (Q5SJH5, Uniprot accession code: Uniprot accession code: RIMM_THET8), d, BRP (P17493, Uniprot accession code: BLE_STRHI) and e, sfGFP (pdb) proteins with alkyne handle biosynthetically incorporated using genetic code expansion technology and pPrF amino acid. The introduced modifications were monitored by MALDI MS of intact proteins in combination with trypsin digestion analysis (DA) which identified peptides with alkyne or NH-triazole fragments at the corresponding positions. In Cu-catalyzed reactions of BRP pPrF (5) and sfGFP pPrF (8) proteins, deamidations of Asn and Gin residues were observed, as common for these fragments under acidic conditions, contributing to a difference of 5 and 6 Da, respectively, between calculated and observed MW*. Comparison of the CD spectra of wild-type and NH-triazole-functionalized RimM (3) and BRP (6) proteins showed that the reaction conditions did not affect their secondary structure.

[0023] Figure 3. a, Reactions for preparation of NH-triazole functionalized native proteins, b, Analysis of native proteins, and their products c, after alkynylation, d, and copper catalyzed reactions, based on MALDI MS. For copper catalyzed reactions, d, due to several alkyne fragments present, and to ensure quantitative conversions, the reactions were conducted for prolonged time (20 h) at elevated temperature (40 °C). Control experiments showed that the reaction did not occur in the absence, as well as in the presence of a catalytic amount of copper (20 mol% of copper to protein). In the cases where no THPTA ligand was used or the reaction was carried out at pH 7, there was no P481445PC00 or very low conversion with the complex mixture formed. We found that the removal of copper ions with EDTA after the reaction is crucial to prevent precipitation of the proteins. The yield of the reaction was determined by measuring the concentration of a conjugated protein mixture after the residual small molecules had been removed by spin filtration. The extinction coefficient for the studied structural fragments, i.e. alkyne handle and NH-triazole, were determined on 4-dodecyl- lH-l,2,3-triazole and 1-pentyne, and found to be 20 and 1 M1cm’1, respectively, contributing very little to the overall extinction coefficient, e, Conformation analysis of proteins 10-22 using CD spectra measurement after reactions and thermal treatment.

[0024] Figure 4: a, Developed NHS reagents for the rapid introduction of NH-triazole into proteins as demonstrated in the case of insulin 10 to produce insulins 19, 27-29 with incorporated NH-triazoles at different linker lengths and ratios, including rapid access to insulin 19 with increased stability, b, MS-MS analysis reveals NH-triazole conjugation sites in 19. The residues of peptides where PEG1- NH-triazole fragment was detected are marked in dark color. There was a very small or unobserved amount of native chain B, which is therefore most likely to be conjugated first, and a small amount of native chain A. Peptides that would correspond to four-fold modified protein, to some extent observed by MALDI-MS (Fig. 3d), were not observed in MS / MS analysis. There are potentially three possible 2-fold conjugated versions, in which Al-Gly and Bl-Phe; or Bl-Phe and B29-Lys; or Al-Gly and B29-Lys, are modified. The latter is the least likely, as no unmodified native B chain was detected in the analysis, b, SEC chromatograms of native 10 and modified insulin 19 at 50 pM, in which the ratio of monomeric-dimer and hexameric (and higher) compositions is seen. Modified insulin forms a significant amount of hexameric state even in the absence of Cu2+ions, whereas native insulin requires 100 pM of Cu2+ions to form a significant amount of hexameric state - at this concentration, modified insulin 19 forms higher order aggregates / polymers. c, Circular dichroism (CD) spectra of native 10 (left) and modified insulin 19 (right) before and after heating to 95 °C. The heating was performed by 1 °C / min program from 25 °C to 95 °C in approximately 90 minutes (CD spectra at single wavelength). In between CD spectra were recorded, which extended the exposure time at the respective temperature. The conformation of the modified insulin 19 is preserved, while the native counterpart precipitates and decomposes. Below left is the photo of CD vials with native 10 (left), forming aggregates, and modified 19 (right), which remain in solution after being heated to 95 °C at a concentration of 50 pM. Comparison of the stability of native 10 and NH-triazole- functionalized 19 insulin at 70 °C over time (bottom right), as estimated by HPLC-MS analysis from protein concentration in solution. The native insulin starts to decompose after 2 hours at 70 °C, P481445PC00 whereas the modified insulin only starts decomposing after 8 hours, d, (i) snapshots of molecular dynamics study, e, Comparing chemical stability of 10 and 19 in the presence of IDE (insulin degrading enzyme). The reactions rates were determined by monitoring concentrations of 10 and 19 over time by HPLC, x-axis; t (min); y-axis: % of degradation as Cdeg / o, Cde = c0-cx

[0025] Figure 5. Serum-starved L6 myotubes were treated (a-d) with 100 nM 10, 19, 19 (95 °C), 19 (60 °C), or rhlns (15 min), (e-h) 25-300 nM 10 or 18 or 100 nM rhlns (15 min), (i-l) 300 nM 10, 19 (5-60 min) or 100 nM rhlns (15 min), or (m) 300 nM 10, 19, 19 (95 °C), 19 (60 °C), or 100 nM rhlns (15 min). Immunoblotting was used to measure (a,e,i) phospho-Akt (Ser473), (b,f,j) phospho-AS160 (Th r642), (c,g,k) phospho-GSK-3a / p (Ser21 / 9), and (d,h,l) phospho-S6RP (Ser235 / 236). Results are means with SEM (n = 4 for a-d and n = 8 for e-l). *p < 0.05 vs. Basal (1-way ANOVA, Dunnett post- hoc test), #p < 0.05 18 vs. 9 in all dose- or time-points (1-way ANOVA, Sidak post-hoc test). Molecular weight (kDa) markers are indicated on the right side of blots. AU = arbitrary units, m, 2- deoxy-glucose uptake. Results are means with SEM (n = 6). *p < 0.05 vs. Basal (1-way ANOVA, Dunnett post-hoc test), n, A simplified scheme of insulin signaling. S6RP: S6 ribosomal protein, GSK- 3a / : glycogen synthase kinase-3 a / , AS160: Akt substrate of 160 kDa, GLUT1 / 4: glucose transporter type 1 / 4, P: phosphorylation, solid arrow: direct effect, dashed arrow: indirect effect.

[0026] Figure 6. Concept of protein purification by pH-tuned affinity chromatography, (i, ii) Schematic representation of separation of 1:1 mixture of 10 and 19 using Cu2+ affinity column and appropriate buffer, the rationale for which is based on (in)ability of imidazole and NH-triazole to bind to metal ions under acidic conditions (iii, iv).

[0027] Description

[0028] Polypeptides of the invention.

[0029] The present invention provides a polypeptide comprising a NH-1, 2, 3, -triazole.

[0030] In some embodiments the NH-l,2,3-triazole is attached to the remaining part of the polypeptide at the carbon in position 4 or 5.

[0031] In some embodiments the NH-l,2,3-triazole has the structure and attachment to the polypeptide R as in any one of Formulae 1-1 to 1-V or a mixture thereof : P481445PC00

[0032] Formulae l-l to 1-V

[0033] In some embodiments the NH-l,2,3-triazole has the structure and attachment to the polypeptide R as in any one of Formulae 1-1 to l-lll or a mixture thereof.

[0034] In some embodiments the NH-l,2,3-triazole has the structure and attachment to the polypeptide as in any one of Formulae 2-1 to 2-V or a mixture thereof :

[0035] Formulae 2-I to 2-V In some embodiments the NH-l,2,3-triazole has the structure and attachment to the polypeptide R as in any one of Formulae 2-I to 2-III or a mixture thereof.

[0036] In some embodiments the NH-l,2,3-triazole is part of the non-proteogenic alpha amino acid residue of any one of Formula 3-I to 3-V or a mixture thereof :

[0037] Formulae 3-I to 3-V

[0038] In some embodiments the NH-l,2,3-triazole is part of the non-proteogenic alpha amino acid residue of any one of Formula 3-I to 3-III or a mixture thereof. P481445PC00

[0039] In some embodiments the NH-l,2,3-triazole is part of the non-proteogenic alpha amino acid residue

[0040] NH-l,2,3-triazole - LI - CHNH2COOH, wherein LI is a linker.

[0041] In some embodiments LI is an optionally substituted Ci-25-alkyl.

[0042] In some embodiments LI is an optionally substituted linear Ci-25-alkyl.

[0043] In some embodiments LI is a Ci-25-alkyl.

[0044] In some embodiments LI is a linear Ci-25-alkyl.

[0045] In some embodiments LI is selected from the group consisting of methyl, ethyl, propyl and n-butyl.

[0046] In some embodiments LI comprises a polyethylene (PEG) moiety.

[0047] In some embodiments the PEG moiety is polydisperse.

[0048] In some embodiments the PEG moiety is monodisperse.

[0049] In some embodiments the PEG moiety has a molecular weight of less than 4000 kDa, less than 2000 kDa or less than 1000 kDa.

[0050] In some embodiments the PEG moiety has the structure -(CH2-0-CH2)n- wherein n is from 1 to 50, from 1 to 25 or from 2 to 15.

[0051] In some embodiments n is 6, 8 or 10.

[0052] In some embodiments the NH-l,2,3-triazole is attached to the epsilon-amino group of a lysine or arginine residue.

[0053] In some embodiments the NH-l,2,3-triazole is attached to the epsilon-amino group of a lysine residue.

[0054] In some embodiments the NH-l,2,3-triazole is attached to the N-terminal amino group of the polypeptide.

[0055] In some embodiments the NH-l,2,3-triazole has replaced an imidazole of a histidine residue of the polypeptide in its naturally occurring form.

[0056] In some embodiments the polypeptide comprises more than one NH-l,2,3-triazole. P481445PC00

[0057] In some embodiments the polypeptides is a derivative of human insulin, desB30-human insulin, or a human insulin analogue.

[0058] In some embodiments the polypeptide is a derivative of human insulin, desB30-human insulin, or a human insulin analogue comprising a NH-l,2,3-triazole attached to B29-Lys.

[0059] In some embodiments the polypeptide is a derivative of human insulin, desB30-human insulin or a human insulin analogues comprising a NH-l,2,3-triazole attached to Bl-Phe.

[0060] In some embodiments the polypeptide is a derivative of human insulin, desB30-human insulin or a human insulin analogues comprising a NH-l,2,3-triazole attached to Al-Gly.

[0061] In some embodiments the polypeptide is human insulin or desB30 human insulin having three NH- 1,2,3-triazoles attached to Al-Gly, Bl-Phe and B29-Lys, respectively.

[0062] In some embodiments the polypeptide is human insulin being or DesB30-human insulin derivatized with a structure comprising a NH-l,2,3-triazole at B29-Lys, Bl-Phe or Al-Gly.

[0063] In some embodiments the polypeptide is human insulin or DesB30-human insulin being derivatized with a structure comprising a NH-l,2,3-triazole at the position : B29-Lys and Bl-Phe, B29-Lys and Al-Gly or Bl-Phe and Al-Gly.

[0064] In some embodiments the polypeptide is a NH-1, 2, 3, -triazole functionalized variant of human insulin, ribosome maturation factor (RIMM), bleomycin resistance protein (BRP), green fluorescent protein (GFP), superfolder GFP (sfGFP) or avidin.

[0065] In some embodiments the polypeptide is a NH-1, 2, 3, -triazole functionalized variant of an antibody.

[0066] In some embodiments the polypeptide is a NH-1, 2, 3, -triazole functionalized variant of an antibody which is trastuzumab.

[0067] In some embodiments the polypeptide is a GLP-1 agonist.

[0068] In some embodiments the polypeptide is a GLP-1 peptide derivative.

[0069] In some embodiments the polypeptide is a NH-1, 2, 3, -triazole functionalized variant of dulaglutide, exenatide, semaglutide, liraglutide or lixisenatide.

[0070] In some embodiments the polypeptide is formed by introducing an alkyne and performing a copper catalyzed cycloaddition of hydrogen azide to the alkyne. P481445PC00

[0071] In some embodiments the polypeptide is formed by bioconjugation of one or more basic protein sites by alkyne-functionalized N-hydroxysuccinimide (NHS) ester followed by copper catalyzed cycloaddition of hydrogen azide to the alkyne.

[0072] In some embodiments the introduction of an alkyne is made by genetic code expansion technology.

[0073] In some embodiments basic residues of the protein is reacted with NH-l,2,3-triazole functionalized NHS ester.

[0074] Methods for synthesis.

[0075] The present invention further provides two different synthesis routes for preparing the polypeptides of the invention.

[0076] Accordingly, in a second aspect the present invention provides a method for preparing the polypeptide as defined above, comprising the steps : a) providing the protein to be NH-l,2,3-triazole functionalized, b) introducing at least one alkyne into the protein to form an alkyne-functionalized protein, c) reacting the alkyne functionalized protein with hydrogen azide in the presence of a copper catalyst, and c) isolating said polypeptide.

[0077] The terms "polypeptide" and "protein" as used herein are both intended to mean an oligomer or polymer of alpha amino acid residues joined by amide bonds. The alpha amino acid residues may be selected from the genetically encode amino acids, from other natural amino acids as well as from derivatives thereof, or from synthetic amino acid residues. The two terms polypeptide and protein are both used in the present text, and generally the language intends to use both terms in the context of a "polypeptide" referring to the NH-l,2,3-triazole functionalized protein, and the term "protein" referring to the molecule used for attaching the NH-l,2,3-triazole functionalization.

[0078] In one embodiment, in step b) the at least one alkyne is introduced by bioconjugation of one or more basic protein sites by alkyne-functionalized NHS ester.

[0079] In another embodiment, in step b) the at least one alkyne is introduced by genetic code expansion technology. P481445PC00

[0080] In yet another embodiment, in step c) the copper catalyst is introduced as a Cu(ll) or Cu ( I ) salt with addition of an ascorbate salt.

[0081] In some embodiments, in step c), a CuAAC-accelerating ligand, e.g. THPTA, is added.

[0082] In some embodiments, in step c) the copper catalyst is CuSO4 in combination with sodium ascorbate and water-soluble CuAAC-accelerating ligand THPTA.

[0083] In some embodiments the reaction is conducted in a buffer at a pH in the range from 1.0 to 8.0, and at a temperature in the range from 10 °C to 90 °C.

[0084] In some embodiments the reaction is conducted in a buffer at a pH in the range from 4.0 to 8.0, and at a temperature in the range from 10 °C to 90 °C.

[0085] In some embodiments the reaction is conducted at a pH in the range from 4.0 to 6.0.

[0086] In some embodiments the reaction is conducted at a temperature in the range from 10 °C to 35 °C.

[0087] In some embodiments the reaction is conducted at a pH of about 5 and at room temperature.

[0088] In a third aspect the present invention provides a method for preparing a polypeptide as defined above, comprising the steps : c) providing the protein to be NH-l,2,3-triazole functionalized, d) providing a NH-l,2,3-triazole functionalized NHS ester, c) reacting the protein with NH-l,2,3-triazole functionalized NHS ester, and d) isolating said polypeptide.

[0089] In one embodiment, in step b) the NH-1, 2, 3, -triazole functionalized NHS ester has the structure:

[0090] NHS-L2-NH-triazole, wherein NHS is N-hydroxy-succinimide and the linker L2 is selected from the group consisting of Ci- 25-alkyl, linear Ci-25-alkyl, optionally substituted Ci-25-alkyl, optionally substituted linear C1-25- alkyl, methyl, ethyl, propyl, n-butyl, and polyethylene (PEG) moiety.

[0091] In another embodiment, in step b) the NH-l,2,3-triazole functionalized NHS ester is selected from compounds 23-26 P481445PC00

[0092] 2,5-dioxopyrrolidin-l-yl lH-l,2,3-triazole-5-carboxylate (23), 2,5-dioxopyrrolidin-l-yl 3- (lH-l,2,3-triazol-5-yl)propanoate (24), 2,5-dioxopyrrolidin-l-yl 3-((lH-l,2,3-triazol-5- yl)methoxy)propanoate (25), 2,5-dioxopyrrolidin-l-yl 3-(2-(2-((lH-l,2,3-triazol-5- yl)methoxy)ethoxy)ethoxy)propa noate (26).

[0093] In another embodiment, in step b) the NH-l,2,3-triazole functionalized NHS ester is prepared by reaction of NH-l,2,3-triazole functionalized carboxylic acid with N,N'-disuccinimidyl carbonate (DSC).

[0094] In another embodiment the reaction of step c) is performed at a temperature from 10 °C to 70 °C, at a pH from 7.1 to 11.0.

[0095] In some embodiments the reaction of step c) is performed at a temperature from 10 °C to 70 °C, at a pH from 7.5 to 9.5.

[0096] In some embodiments the reaction of step c) is performed at a temperature from 60 °C to 70 °C, at a pH from 7.5 to 9.5.

[0097] In some embodiments the reaction in steps c) is performed at a temperature of about 65 °C and a pH of about 8.5.

[0098] In some embodiments in step c) the reaction is performed in a buffer.

[0099] In some embodiments in step c) the reaction is performed in a buffer which is e.g., phosphate buffered saline (PBS), TRIS, phosphate buffers, bicarbonate buffers, protein buffers and Good's buffers .

[0100] Method for stabilization of a protein. P481445PC00

[0101] In a fourth aspect the present invention provides a method for preparing a polypeptide being a stabilized variant of a protein having a certain biological function, which method comprises introducing at least one lH-l,2,3-triazole moiety into said protein, thereby providing a polypeptide having increased stability and substantially the same biological function.

[0102] In one embodiment the polypeptide is as defined in the first aspect of the present invention.

[0103] Method for purifying a polypeptide comprising a lH-l,2,3-triazole functionalization.

[0104] In a fifth aspect the present invention provides a method for purifying a polypeptide which comprises at least one lH-l,2,3-triazole moiety, wherein said method comprises metal affinity chromatography.

[0105] In one embodiment the metal affinity chromatography is Cu2+affinity chromatography, Ni2+affinity chromatography, Co2+affinity chromatography, Zn2+, affinity chromatography or Fe3+ / Fe2+affinity chromatography.

[0106] In one embodiment the metal affinity chromatography is Cu2+affinity chromatography.

[0107] In one embodiment the metal affinity chromatography comprises two-step elution where the first elution is by first eluent being a buffer having a pH from about 3.0 to about 5.0 and where the first eluent does not contain 1,2,3-triazole, and where the second elution is by a second eluent being a buffer having a pH from about 3.0 to about 5.0 and where the second eluent does comprise 1,2,3- triazole.

[0108] In another embodiment the buffer in the first eluent and in the second eluent are both an acetate buffer.

[0109] Pharmaceutical formulations.

[0110] In a sixths aspect the present invention provides a pharmaceutical formulation comprising the polypeptide as defined in the first aspect of the invention, and at least one pharmaceutically acceptable excipient.

[0111] In one embodiment the pharmaceutical formulation is an aqueous formulation. P481445PC00

[0112] Pharmaceutical formulations of the polypeptides comprising a NH-l,2,3-triazole basically have a composition as known for protein formulation in general. The pharmaceutically acceptable excipient may comprise buffer, preservatives, isotonicity agents and the like.

[0113] The following list of items further describe the present invention :

[0114] 1. Polypeptide comprising a NH-l,2,3-triazole.

[0115] 2. The polypeptide according to item 1, wherein the NH-l,2,3-triazole is attached to the remaining part of the polypeptide at the carbon in position 4 or 5.

[0116] 3. The polypeptide according to item 1 or 2, wherein the NH-l,2,3-triazole has the structure and attachment to the polypeptide R as in any one of Formulae 1-1 to 1-V or a mixture thereof :

[0117] Formulae l-l to 1-V

[0118] 4. The polypeptide according to any of the preceding items, wherein the NH-l,2,3-triazole has the structure and attachment to the polypeptide R as in any one of Formulae 1-1 to l-lll or a mixture thereof.

[0119] 5. The polypeptide according to item 1 or 2, wherein the NH-l,2,3-triazole has the structure and attachment to the polypeptide as in any one of Formulae 2-1 to 2-V or a mixture thereof

[0120] Formulae 2-1 to 2-V P481445PC00

[0121] 6. The polypeptide according to any one of items 1-2 and 5, wherein the NH-l,2,3-triazole has the structure and attachment to the polypeptide R as in any one of Formulae 2-1 to 2-111 or a mixture thereof.

[0122] 7. The polypeptide according to any of the items 1-2 and 5-6, wherein the NH-l,2,3-triazole is part of the non-proteogenic alpha amino acid residue of any one of Formula 3-1 to 3-V or a mixture thereof :

[0123] Formulae 3-I to 3-V

[0124] 8. The polypeptide according to any one of items 5-7, wherein the NH-l,2,3-triazole is part of the non-proteogenic alpha amino acid residue of any one of Formula 3-I to 3-III or a mixture thereof.

[0125] 9. The polypeptide according to any of the preceding items, wherein the NH-l,2,3-triazole is part of the non-proteogenic alpha amino acid residue :

[0126] NH-l,2,3-triazole - LI - CHNH2COOH, wherein LI is a linker.

[0127] 10. The polypeptide according to item 9, wherein LI is an optionally substituted Ci-25-alkyl.

[0128] 11. The polypeptide according to item 9 or 10, wherein LI is an optionally substituted linear Ci-

[0129] 12. The polypeptide according to any one of items 9-11, wherein LI is a Ci-25-alkyl.

[0130] 13. The polypeptide according to any one of items 9-12, wherein LI is a linear Ci-25-alkyl.

[0131] 14. The polypeptide according to any one of items 9-13, wherein LI is selected from the group consisting of methyl, ethyl, propyl and n-butyl. P481445PC00

[0132] 15. The polypeptide according to item 9, wherein LI comprises a polyethylene (PEG) moiety.

[0133] 16. The polypeptide according to item 15, wherein the PEG moiety is polydisperse.

[0134] 17. The polypeptide according to item 15, wherein the PEG moiety is monodisperse.

[0135] 18. The polypeptide according to any one of items 15-17, wherein the PEG moiety has a molecular weight of less than 4000 kDa, less than 2000 kDa or less than 1000 kDa.

[0136] 19. The polypeptide according to any one of items 15-18, wherein the PEG moiety has the structure -(CH2-0-CH2)n- wherein n is from 1 to 50, from 1 to 25 or from 2 to 15.

[0137] 20. The polypeptide according to item 19, wherein n is 6, 8 or 10.

[0138] 21. The polypeptide according to anyone of the preceding items, wherein the NH-l,2,3-triazole is attached to the epsilon-amino group of a lysine or arginine residue.

[0139] 22. The polypeptide according to anyone of the preceding items, wherein the NH-l,2,3-triazole is attached to the epsilon-amino group of a lysine residue.

[0140] 23. The polypeptide according to anyone of the preceding items, wherein the NH-l,2,3-triazole is attached to the N-terminal amino group of the polypeptide.

[0141] 24. The polypeptide according to anyone of the preceding items, wherein the NH-l,2,3-triazole has replaced an imidazole of a histidine residue of the polypeptide in its naturally occurring form.

[0142] 25. The polypeptide according to any one of the preceding items, wherein the polypeptide comprises more than one NH-l,2,3-triazole.

[0143] 26. The polypeptide according to any one of the preceding items, which is a derivative of human insulin, desB30-human insulin, or a human insulin analogue.

[0144] 27. The polypeptide according to any one of the preceding items, which is a derivative of human insulin, desB30-human insulin, or a human insulin analogue comprising a NH-1,2,3- triazole attached to B29-Lys.

[0145] 28. The polypeptide according to any one of items 25-27, which is a derivative of human insulin, desB30-human insulin or a human insulin analogues comprising a NH-l,2,3-triazole attached to Bl-Phe. P481445PC00 The polypeptide according to any one of items 25-28, which is a derivative of human insulin, desB30-human insulin or a human insulin analogues comprising a NH-l,2,3-triazole attached to Al-Gly. The polypeptide according to any one of items 25-29, which is human insulin or desB30 human insulin having three NH-l,2,3-triazoles attached to Al-Gly, Bl-Phe and B29-Lys, respectively. The polypeptide according to any one of items 25-30, which is human insulin being or DesB30-human insulin derivatized with a structure comprising a NH-l,2,3-triazole at B29- Lys, Bl-Phe or Al-Gly. The polypeptide according to any one of items 25-31, which is human insulin or DesB30- human insulin being derivatized with a structure comprising a NH-l,2,3-triazole at the position : B29-Lys and Bl-Phe, B29-Lys and Al-Gly or Bl-Phe and Al-Gly. The polypeptide according to any one of items 1-25, which is a NH-1, 2, 3, -triazole functionalized variant of human insulin, ribosome maturation factor (RIMM), bleomycin resistance protein (BRP), green fluorescent protein (GFP), superfolder GFP (sfGFP) or avidin. The polypeptide according to any one of items 1-25, which is a NH-1, 2, 3, -triazole functionalized variant of an antibody. The polypeptide according to item 34, wherein the antibody is trastuzumab. The polypeptide according to any one of items 1-24, which is a GLP-1 agonist. The polypeptide according to item 36, which is a GLP-1 peptide derivative. The polypeptide according to item 36 or 37, which is a NH-1, 2, 3, -triazole functionalized variant of dulaglutide, exenatide, semaglutide, liraglutide or lixisenatide. The polypeptide according to any one of the preceding items, which is formed by introducing an alkyne and performing a copper catalyzed cycloaddition of hydrogen azide to the alkyne. The polypeptide according to any one of the preceding items, which is formed by bioconjugation of one or more basic protein sites by alkyne-functionalized N- P481445PC00 hydroxysuccinimide (NHS) ester followed by copper catalyzed cycloaddition of hydrogen azide to the alkyne.

[0146] 41. The polypeptide according to any one of items 1-39, where the introduction of an alkyne is made by genetic code expansion technology.

[0147] 42. The polypeptide according to any one of items 1-39, wherein basic residues of the protein is reacted with NH-l,2,3-triazole functionalized NHS ester.

[0148] 43. Method for preparing the polypeptide as defined in any one of items 1-41, comprising the steps : a) providing the protein to be NH-l,2,3-triazole functionalized, b) introducing at least one alkyne into the protein to form an alkyne-functionalized protein, c) reacting the alkyne functionalized protein with hydrogen azide in the presence of a copper catalyst, and c) isolating said polypeptide.

[0149] 44. The method according to item 43, wherein in step b) the at least one alkyne is introduced by bioconjugation of one or more basic protein sites by alkyne-functionalized NHS ester.

[0150] 45. The method according to item 43, wherein in step b) the at least one alkyne is introduced by genetic code expansion technology.

[0151] 46. The method according to any one of items 43-45, wherein in step c) the copper catalyst is introduced as a Cu(ll) or Cu(l) salt with addition of an ascorbate salt.

[0152] 47. The method according to item 46, wherein in step c), a CuAAC-accelerating ligand, e.g. THPTA, is added.

[0153] 48. The method according to any one of items 43-47, wherein in step c) the copper catalyst is CuSO4 in combination with sodium ascorbate and water-soluble CuAAC-accelerating ligand THPTA.

[0154] 49. The method according to any one of items 43-48, wherein the reaction is conducted in a buffer at a pH in the range from 1.0 to 8.0, and at a temperature in the range from 10 °C to 90 °C. P481445PC00

[0155] 50. The method according to any one of items 43-49, wherein the reaction is conducted in a buffer at a pH in the range from 4.0 to 8.0, and at a temperature in the range from 10 °C to 90 °C.

[0156] 51. The method according to any one of items 43-50, wherein the reaction is conducted at a pH in the range from 4.0 to 6.0.

[0157] 52. The method according to any one of items 43-51, wherein the reaction is conducted at a temperature in the range from 10 °C to 35 °C.

[0158] 53. The method according to any one of items 43-52, wherein the reaction is conducted at a pH of about 5 and at room temperature.

[0159] 54. Method for preparing a polypeptide as defined in any one of items 1-38 and 42, comprising the steps : e) providing the protein to be NH-l,2,3-triazole functionalized, f) providing a NH-l,2,3-triazole functionalized NHS ester, c) reacting the protein with NH-l,2,3-triazole functionalized NHS ester, and d) isolating said polypeptide.

[0160] 55. The method according to item 55, wherein in step b) the NH-1, 2, 3, -triazole functionalized NHS ester has the structure:

[0161] NHS-L2-NH-triazole, wherein NHS is N-hydroxy-succinimide and the linker L2 is selected from the group consisting of Ci-25-alkyl, linear Ci-25-alkyl, optionally substituted Ci-25-alkyl, optionally substituted linear Ci-25-alkyl, methyl, ethyl, propyl, n-butyl, and polyethylene (PEG) moiety.

[0162] 56. The method according to item 54 or 55, wherein in step b) the NH-l,2,3-triazole functionalized NHS ester is selected from compounds 23-26

[0163] P481445PC00

[0164] 2,5-dioxopyrrolidin-l-yl lH-l,2,3-triazole-5-carboxylate (23), 2,5-dioxopyrrolidin-l-yl 3- (lH-l,2,3-triazol-5-yl)propanoate (24), 2,5-dioxopyrrolidin-l-yl 3-((lH-l,2,3-triazol-5- yl)methoxy)propanoate (25), 2,5-dioxopyrrolidin-l-yl 3-(2-(2-((lH-l,2,3-triazol-5- yl)methoxy)ethoxy)ethoxy)propa noate (26). The method according to any one of items 54-56, wherein in step b) the NH-l,2,3-triazole functionalized NHS ester is prepared by reaction of NH-l,2,3-triazole functionalized carboxylic acid with N,N'-disuccinimidyl carbonate (DSC). The method according to any one of items 54-57, wherein the reaction of step c) is performed at a temperature from 10 °C to 70 °C, at a pH from 7.1 to 11.0. The method according to any one of items 54-58, wherein the reaction of step c) is performed at a temperature from 10 °C to 70 °C, at a pH from 7.5 to 9.5. The method according to any one of items 54-59, wherein the reaction of step c) is performed at a temperature from 60 °C to 70 °C, at a pH from 7.5 to 9.5. The method according to any one of items 54-60, wherein the reaction in steps c) is performed at a temperature of about 65 °C and a pH of about 8.5. The method according to any one of items 54-61, wherein in step c) the reaction is performed in a buffer. The method according to any one of items 54-62, wherein in step c) the reaction is performed in a buffer which is e.g., phosphate buffered saline (PBS), TRIS, phosphate buffers, bicarbonate buffers, protein buffers and Good's buffers . P481445PC00

[0165] 64. Method for preparing a polypeptide being a stabilized variant of a protein having a certain biological function, which method comprises introducing at least one lH-l,2,3-triazole moiety into said protein, thereby providing a polypeptide having increased stability and substantially the same biological function.

[0166] 65. Method for purifying a polypeptide which comprises at least one lH-l,2,3-triazole moiety, wherein said method comprises metal affinity chromatography.

[0167] 66. The method according to item 65, wherein the metal affinity chromatography is Cu2+affinity chromatography, Ni2+affinity chromatography, Co2+affinity chromatography, Zn2+, affinity chromatography or Fe3+ / Fe2+affinity chromatography.

[0168] 67. The method according to item 65 or 66, wherein the metal affinity chromatography is Cu2+affinity chromatography.

[0169] 68. The method according to any one of items 65-67, wherein a solution comprising said polypeptide comprising at least one lH-l,2,3-triazole moiety is applied to a metal affinity chromatographic material, and wherein said solution has a pH of less than 6.0.

[0170] 69. The method according to any one of items 65-68, wherein said metal affinity chromatography comprises two-step elution where the first elution is by first eluent being a buffer having a pH from about 3.0 to about 5.0 and where the first eluent does not contain 1,2,3-triazole, and where the second elution is by a second eluent being a buffer having a pH from about 3.0 to about 5.0 and where the second eluent does comprise 1,2,3-triazole.

[0171] 70. The method according to item 69, wherein the buffer in the first eluent and in the second eluent are both an acetate buffer.

[0172] 71. The method according to any one of items 64-70, wherein said polypeptide is as defined in any one of items 1-42.

[0173] 72. Pharmaceutical formulation comprising the polypeptide as defined in any one of items 1- 42 and at least one pharmaceutically acceptable excipient.

[0174] 73. The pharmaceutical formulation according to item 72, which is an aqueous formulation. P481445PC00

[0175] Various examples are described hereinafter with reference to the figures. It should be noted that the figures are only intended to facilitate the description of the examples. They are not intended as an exhaustive description of the claimed invention or as a limitation on the scope of the claimed invention. In addition, an illustrated example needs not have all the aspects or advantages shown. An aspect or an advantage described in conjunction with a particular example is not necessarily limited to that example and can be practiced in any other examples even if not so illustrated, or if not so explicitly described.

[0176] The present examples may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the examples are merely described below, by referring to the figures, to explain aspects.

[0177] Examples

[0178] Example 1 - Site-specific incorporation of NH-triazoles by copper-catalyzed reaction of biosynthetically installed alkyne sites

[0179] Using proteins as substrates in chemical reactions without affecting their structure and function remains a key challenge, given that proteins are conformationally heterogeneous molecules with various unprotected functional groups. Furthermore, preserving their native structure requires very specific reaction conditions, i.e. aqueous media, low reaction temperatures, appropriate pH values, and low (pM) concentrations.'''™ Transition metal-mediated modifications of proteins are, due to their unique reactivity and high selectivity, a rapidly developing field that can address these reaction requirements.1''''''™ ’"' We have previously reported successful incorporation of NH- triazoles into small molecules and peptides via a copper-catalyzed reaction of alkyne handle with hydrogen azide, which could be formed in situ from sodium azide.Xl" We envisioned that similar strategy could be extended to proteins provided sufficiently mild reaction conditions which would not perturb native protein structure. We saw an opportunity in the pKa value of 4.69 of hydrogen azide,XVIwhich can be formed from sodium azide under slightly acidic conditions potentially tolerated by proteins, giving access to sufficient quantity of the key reaction component, i.e. HN3, in aqueous media (Fig. 2a, b).

[0180] To test this hypothesis, we recombinantly expressed two protein substrates with alkyne handle through amber codon suppression,’"'" ’"'1" using orthogonal tRNA / aminoacyl synthetase pairs of a P481445PC00 tyrosine analog with propargyl handle (4-propargyloxy-l-phenylalanine, pPrF) in E. coli expression systems.XIXIn this way, modified versions of N-terminal domain of the ribosome maturation factor (RimM, 1, Fig 2c)xxfrom the thermophilic organism Thermus thermophilus, a bleomycin resistance protein (BRP, 4, Fig. 2d) from Streptoalloteichus hindustanus,XXIwhich provides antibiotic resistance, and superfolder GFP (sfGFP, 7, Fig. 2e) were prepared. In RimM (1), the substitution of Tyr34 by pPrF resulted in a surface-exposed alkyne (RimM pPrF, 2), while in BRP (4) the substitution of Try75 by pPrF the alkyne handle is less exposed (BRP pPrF, 5), allowing the investigation of steric effects on the CuAAC reaction (Fig. 2c, d). Substitution of Asnl50 with pPrF in sfGPF 7 smiliray resulted in sfGPF with surface exposed pPrF (8).

[0181] When screening different reaction conditions, we found that using CuSCU, in combination with sodium ascorbate (Na(asc)) and water-soluble CuAAC-accelerating ligand THPTA, and aqueous pH 5 buffer at room temperature (Fig. 2a) yielded RimM (3)-, BRP (6)- and sfGFP (9) NH-triazole- functionalized proteins in quantitative conversion (Fig. 2c-e).

[0182] It is noteworthy that for a successful reaction, all reagents are required in the corresponding equivalent to the protein, whereby several processes must take place for a successful outcome. (Fig. 2a, b). Namely, a suitable pH value, e.g. 5, that is still tolerated by the proteins and ensures sufficient in situ formation of hydrogen azide from sodium azide is key for the successful outcome (Fig. 2b, i). In addition to the copper ions and the THPTA ligand, which form the catalytic system (Fig. 2b, iii), the presence of Na(asc) is also crucial for a successful reaction. Na(asc) provides the reducing conditions required for Cu( I ) formation and at the same time prevents oxidative damage to proteins by reactive oxygen species (ROS), which usually form in the presence of copper, and thus has a dual function (Fig. 2b, ii).xx"'xxl" in the absence of Na(asc) or at a higher pH of 7, the reaction proceeded to small extent to the products, but the resulting protein mixtures were complex, in addition substantial amount of precipitate formed in the absence of Na(asc). In the absence of copper, however, the reaction did not take place.

[0183] For proteins with a recombinantly expressed alkyne handle, like RimM pPrF 2 and BRP pPrF 5 (Fig. 2c, d), the reaction proceeded rapidly even in the absence of the THPTA ligand. This very interesting observation suggests the acceleration of the reaction by the protein surface, since the reactions with alkynes conjugated to the proteins and whose alkyne is further away from the protein surface (see Fig. 3 below) did not proceed in the absence of THPTA. The same is the case for small molecule substrates, where the reactions, although carried out at much higher concentrations (up to 20,000- P481445PC00 fold higher; 0.8 M vs 40 pM), depend on the presence of CuAAC-accelerating ligands such as TBTA and do not proceed to NH-triazole products in their absence at temperatures below 80 °C.XI"

[0184] Considering that the reactions were carried out at low protein concentrations (20-40 pM), at room temperature and proceeded to the products with 62% conversion or higher (except in the case of 4 in the absence of THPTA) within 1 min (Fig. 2c-e), they exhibited very rapid kinetics similar to the rates of the click reaction. The proteins tolerated the reaction conditions, as the NH-triazole- functionalized 3 and 6 were isolated in high yields and retained their secondary structure, as evidenced by comparison of their far-UV CD spectra with the native counterparts (Fig. 2c, d). Overall, this demonstrates that the described copper-based catalysis with recombinantly expressed alkyne sites in proteins allows site-specific incorporation of NH-triazole under native-like reaction conditions without perturbing protein native structure.

[0185] Example 2 - Direct bioconjugation of basic protein sites by employing NHS esters

[0186] Despite enabling site-specific incorporation and their homogeneous form, it is more difficult to biosynthesize modified proteins than to obtain them in accessible native form. We therefore aimed to apply the developed reaction to native proteins 10-12 by pre-installing alkyne handle(s) using bioconjugation of native NH2amine groups with NHS esters 13, 14 (Fig. 3a). To test this hypothesis, we selected three proteins with different structural properties and functions (Fig. 3b), i.e., glucose- regulating hormone insulin 10,xxlvavidin 11, a tetrameric protein that forms one of the strongest non-covalent interactions with biotin in nature, used as a probe for a variety of applications in biochemical assays, diagnostics, affinity purification and drug delivery ™ and the clinically used monoclonal antibody trastuzumab 12 used for treatment of breast and stomach cancer.XXVI

[0187] First, we investigated the bioconjugation reaction of native NH2groups (lysine, N-terminal sites) with alkyne-functionalized NHS esters to produce alkyne-functionalized proteins that would serve for the subsequent copper-catalyzed reaction with HN3 (Fig. 3c). The reactions were carried out at protein concentrations of 10 to 172 pM, at room temperature in a pH 7.4 PBS buffer for 1 h, with 10 or 100 equivalents of 12 or 13. The number of fragments bound to the protein in 15-18 was monitored by Matrix-assisted laser ionization mass spectrometry mass spectrometry (MALDI-MS). We were able to conjugate 3 alkynes with different linker lengths to the insulin in 15 and 16 using P481445PC00 either 13 or 14 (Fig. 3c, i). Similarly, by using 14 6 and 32 alkynes were conjugated to avidin and trastuzumab in 17 (Fig. 3c, ii) and 18 (Fig. 3c, iii), respectively.

[0188] With the alkyne-functionalized proteins in hands 15-18, we performed the developed copper- catalyzed reaction (Fig. 3d). The optimized reaction protocol resulted in complete conversion of the alkyne handles to the NH-triazole functionalized proteins in the case of insulins 19 and 20, and almost complete conversion in the case of avidin 21 and trastuzumab 22. In all cases 19-22, heterogeneous mixtures of proteins were formed. With smaller insulins, close monitoring of the reaction was possible, and in reaction producing 20, in which alkynes were attached by PEG5, gave protein in a surprisingly homogeneous form with three NH-triazoles (Fig. 3d, ii). On the other hand, a more heterogenous mixture of insulin 19 with PEG1 linker was observed (Fig. 3d, i). By the procedure described we were able to attach 4 and 26 NH-triazoles to the surface of modified avidin 21 (Fig. 3d, iii) and trastuzumab 22 (Fig. 3d, iv), respectively.

[0189] The effect of NH-triazole modifications on protein secondary structure and thermal stability was investigated using CD spectrometry (Fig. 3e). The conformations of the alkyne- and NH-triazole- modified proteins were largely conserved compared to the unmodified proteins in almost all cases (Fig. 3e, i-iv). The CD spectra of insulins with 3-covalently bound alkynes 15 or NH-triazoles 19 via PEG1 linker showed only minor structural changes compared to the wild type 10 (Fig. 3e, i). On the other hand, the most noticeable conformational change was observed for insulins with a longer PEGylated chain, in both cases with attached three alkynes in 16 or NH-triazoles in 20 (Fig. 3e, ii). For the larger proteins, i.e. avidin 11 and trastuzumab 12, CD spectra of the wild-type proteins almost completely matched their modified versions 17 and 21 (Fig. 3e, iii), and 18 and 22 (Fig. 3e, iv).

[0190] In the case of trastuzumab, a slight thermal stabilization was observed in both modified versions 18 and 22 (Fig. 3e, viii). In case of avidin, the melting points of alkynylated 17 and NH-triazolated 21 avidins were comparable and significantly higher (dTm=15 °C) than that of the wild type 11 (Fig. 3e, vii). Since no difference was observed between the alkyne and NH-triazole variants, the stabilization is probably due to the PEGylation of a protein, which is known to increase thermal stability.xxv"-xx' / l" However, in the case of insulin, a significant increase in thermal stability was observed when three NH-triazoles were attached by PEG1 linker (19) (Fig. 3e, v). While it was not possible to determine the exact melting point of insulin due to the low cooperativity of the transition, the modifications clearly affected denaturation reversibility and / or prevented precipitation. Triazole-modified insulin P481445PC00

[0191] 19 remained in solution after being heated to 95 °C with a CD spectrum comparable to that before thermal treatment (Fig. 3e, v). Its analogue 15 with three alkynes, as well as all other insulins within the study, i.e. 10, 16, 20, precipitated from solution during heating to 95 °C (Fig. 3e, vi). This indicated stabilization by the introduced NH-triazoles, whereby the appropriate length of the linker was also playing a decisive role, since insulin 20 with three NH-triazoles attached via PEG5 linker decomposed.

[0192] Example 3 - NH-triazole NHS esters enable rapid and efficient NH-triazole bioconjugation of human insulin

[0193] Having observed that NH-triazoles can significantly improve protein stability our goal was to develop a rapid, one-step and robust protocol for NH-triazole functionalization of native proteins. For this, the reaction of native insulin 10 with NH-triazole-functionalized NHS ester seemed the obvious choice (Fig. 4a). We prepared several NH-triazole functionalized NHS esters 23-26 by reactions of NH-triazole-functionalized carboxylic acids with A / ,A / '-disuccinim idyl carbonate (DSC), to enable modular incorporation of NH-triazole via basic protein residues (Fig. 4a, top). Optimization of the reaction conditions enabled the preparation of 19 in a 1 hour at 60 °C in Tris pH 8.5 buffer from wild-type 10 in 85% isolated yield on 1 milligram scale. Under the same reaction conditions, other developed NH-triazole NHS reagents 23-26 were also applied to 10, resulting in different conjugation efficiencies depending on the length of the linker (Fig. 4a, bottom). Importantly, these novel NHS reagents are water-soluble and can potentially be used for the rapid incorporation of NH-triazoles not only into proteins but also into various structures, e.g. small molecules, materials, polymers, as is common with NHS esters.

[0194] The reaction of NHS reagent 25 with wild-type insulin 10 (Fig. 4a) resulted in the same heterogeneous insulin 19 as the protocol with initial conjugation of the alkyne handles followed by a copper-catalyzed reaction (Fig. 3c). To clearly determine the conjugation sites in 19, MS / MS analysis was performed, which revealed conjugation at Al-Gly, Bl-Phe and B29-Lys (Fig. 4b). In addition to the main 3-fold conjugated product, significant amounts of 2-fold and small amounts of 4-fold conjugated insulins were observed with MALDI-MS (Fig. 3d see MALDI-MS spectrum for 19).

[0195] In a recent study, phenylalanine-B29-Lys-conjugated insulin was reported to be thermally stable at high temperatures (65 °C).XXIXIt was found that both the aromatic ring of phenylalanine for n-n interactions with A19-Tyr and the presence of the amine group for hydrogen bonding are required for the described stabilization effect.XXIXIt is noteworthy that the same interactions, i.e. n-n and P481445PC00 hydrogen bonding, are both provided by the NH-triazole conjugated at B29-Lys. The significantly higher thermal stability of insulin 19 compared to the described phenylalanine-B29-Lys conjugate could also be a consequence of the additional A / H-triazoles at the N-termini, providing additional interactions, as suggested by the molecular dynamics.

[0196] Example 4 - NH-triazole modification increases thermal and proteolytic stability of insulin as well as its affinity for copper ions

[0197] With growing number of protein and antibody-based biopharmaceuticals, strategies to improve their thermal and chemical stability are becoming increasingly important.XXX'XXXI'XXX"'XXXI" Insulin,XXXIVthe first small protein used in the clinic,™" is a representative example of the complications arising from protein instability, as it is dependent on the cold chain during production, transportation and storage.xxxvl'xxxv"'xxx' / l" Several approaches were attempted to increase its stability and prevent aggregation,XXXVI-XXXV" with the most applied reversible formation of a hexameric state using metal ions through BlO-His coordination.xxxv" The covalent introduction of steric hindrances, another common approach to stabilize biologies,XXXIXsuch as by pegylation of the monomeric form at B29- Lys.xlThere are also several reports of insulin analogues with a redesigned amino acid sequence, among others, to introduce an additional disulfide bond / 1' or even to produce evolutionary- inspiredxl" de novo single-chain jnsu lin .|VI'XXXVI'XXXV" Stabilization attempt by covalently linking peptide chains via the C-terminal Al-Gly and the B29-Lys,xh" unfortunately resulted in inactive insulin, which is conformation-dependent needing the ability to separate the N-terminal of A-chain and the C-terminal of B-chain for interaction with the insulin receptor.X|IV

[0198] Native insulin is usually present in equilibrium between monomeric and dimeric structure.xlvThe stability of insulin is strongly dependent on its quaternary structure, with hexameric form, usually induced by addition of Cu2+or Zn2+ions, being the most stable.X|VIThe self-assembly of histidine- rich peptides and proteins is of crucial importance in biology, as the histidine moiety is both a multifunctional regulator and an ideal motif for the assembly of complex biological structures.''1''" Similarly, 1,4-disubstituted 1,2,3-triazole, product of typical CuAAC,xl''"'xl''"l xllxwhich can act as both a hydrogen donor and acceptor due to its large dipole moment,1 11was the central part of oligomers with induced hybrid helixes to mimic self-assembly in Therefore, the high impact of the introduced NH-triazoles on the protein structure, especially for smaller proteins, as shown in the case of insulin 19, is not surprising. P481445PC00

[0199] The modified insulin 19 is already present in hexameric form in the absence of metal ions, similarly to single-chain insulin.|VI19 showed a high affinity for copper ions compared to the native form 10, as shown by size-exclusion chromatography (SEC) analysis (Fig. 4b, bottom). At 50 pM in the protein, the presence of 1 pM Cu2+ions already induced formation of the hexameric form in approximately 30% in the case of 19, whereas in the case of wild type 10 a 100-fold higher concentration of Cu2+ions was required for a similar effect at the same protein concentration.

[0200] We aimed to explore this affinity for copper ions by purifying a mixture of 10 and 19 on a copper column, which shows another potential application of NH-triazole and developed NHS reagents for their rapid introduction to enhance protein affinity for metal ions.

[0201] A further analysis of the thermal stability of the prepared insulin 19 by recording the CD spectra of native 10 and modified insulin 19 before, immediately after and 16 hours after thermal treatment (Fig. 4c, top) indeed showed a significant increase of thermal stability compared to the wild type 10. The conformation of the modified insulin 19 remained intact after heating to 95 °C, whereas the native insulin decomposed, as evidenced by CD and HPLC-MS analysis, and precipitated out of solution (Fig. 4c, bottom left). A similar observation was made when we monitored the stability of the modified insulin 19 compared to the native counterpart 10 when heated in PBS pH 7.4 at 70 °C over time (Fig. 4c, bottom right).

[0202] The chemical stability towards the insulin degrading enzyme ( I D E)lv" was also increased in the case of 19 compared to native 10. Comparison of the initial decomposition rates in the presence of 4 and 2 mol% IDE revealed a 1.6- and 1.4-fold increase in the stability of 19, respectively. The increased stability was also evident in the remaining insulin concentration over a longer period (24 hours), where the native insulin 10 and modified insulin 19 degraded by 95 and 65% respectively.

[0203] Example 5 - NH-triazole modified insulin retains its biological activity

[0204] Since we did not alter the pharmacophore region of insulin in 19 and it is stabilized by non-covalent interactions, we were interested in its biological activity and whether it is retained after exposure to extreme temperature (95 °C for 2 min). To determine this, L6 myotubes were treated with 100 nM 10, 19 (without or with thermal treatment at 95 °C [19 (95 °C)], as described in Fig. 4., or 3 days (72 h) at 60 °C [19 (60 °C)]), or recombinant human insulin for human use (rhlns) for 15 min. The modified insulins 19, 19 (95 °C), and 19 (60 °C) increased the phosphorylation (Ser473) of Akt P481445PC00

[0205] (aka protein kinase B, PKB) (Fig. 5a), a major kinase downstream of the insulin receptor, indicating the insulin receptor and Akt were activated. Consistent with activation of the Akt / PKB pathway (Fig. 5n), 19, 19 (95 °C), and 19 (60 °C) increased also the phosphorylation (Thr642) of the Akt substrate of 160 kDa (AS160) (Fig. 5b), a Rab GTPase activating protein that regulates the translocation of glucose transporters (GLUT4) to the cell membrane. Also, the phosphorylation (Ser21 / 9) of glycogen synthase kinase-3a / (GSK-3a / ) was observed (Fig. 5c), another direct substrate of Akt, and the phosphorylation (Ser235 / 236) of ribosomal protein S6 (S6RP) (Fig. 5d), a downstream effector of the mammalian target of rapamycin (mTOR) pathway whose activity is promoted by Akt. These results indicated that 19, 19 (95 °C), and 19 (60 °C) were biologically active and mimicked effects of rhlns on the Akt / PKB signaling pathway.

[0206] Concentration dependency was assessed by treating L6 myotubes with 25-300 nM 19 or 10 for 15 min. The phosphorylation of Akt (Fig. 5e), AS160 (Fig. 5f), GSK-3a / (Fig. 5g), and S6RP (Fig. 5h), which was increased by 19 and 10 already at 25 nM, was clearly concentration-dependent. While 19 was less potent inducer of Akt phosphorylation than 10, differences in the response of downstream effectors (Fig. 5f— h) were minor. Time dependency was evaluated by treating L6 myotubes with 300 nM 19 and 10 for 5-60 min. 19 was somewhat less effective inducer of Akt phosphorylation than 10 (Fig. 5i) but nevertheless potently increased the phosphorylation of AS160 (Fig. 5j), GSK-3a / p (Fig. 5k), and S6RP (Fig. 51). These results indicated that NH-triazole- functionalized insulin 19 activated the Akt / PKB pathway in a concentration- and time-dependent manner that is very similar to the effects of native insulin 10.

[0207] To determine whether activation of the Akt / PKB signaling pathway by 19 translates into a physiological response, transmembrane transport of glucose was assessed (Fig. 5n). As estimated by measuring the uptake of3H-2-deoxyglucose in L6 myotubes, a 15-min treatment with 300 nM 19, 19 (95 °C), and 19 (60 °C) stimulated glucose uptake to a similar extent as 300 nM 10 or 100 nM rhlns (Fig. 5m). Taken together, these results show that 19 not only mimics effects of native insulin, but that its biological activity is not lost upon heating.

[0208] Example 6 - Cu2+ affinity purification of NH-triazole-functionalized human insulin.

[0209] NH-triazole binds metal ions across the whole pH range, e.g. the observed significantly increased affinity of 19 for copper ions, unlocking pH-tuned affinity purification of proteins (Fig. 6). The native P481445PC00 form of insulin binds to divalent metal ions under neutral conditions via the BlO-His residue. We hypothesized that the histidine's imidazole is protonated under acidic conditions (below pH 6), which would impair its ability to chelate metal ions, whereas this is not the case for the NH-triazole fragment, which could still interact with d-block metals under such conditions. To test whether the NH-triazole-functionalized human insulin 19 can be selectively separated from its native form 10, we performed a copper affinity chromatographic separation of a 1:1 mixture of 10 and 19 at 35 pM. When using pH 7.4 histidine buffer as eluent, both insulins eluted together. However, when we used pH 4 acetate buffer as the elution buffer, the native insulin eluted first, as shown by HPLC-MS of the first chromatographic peak. We continued the elution with pH 4 buffer and added 1H-1,2,3- triazole as an additive, which eluted the modified insulin 19. This shows that NH-triazole modified proteins can be selectively purified using metal-affinity chromatography, also in presence of a competing interactions via histidine residues.

[0210] Example? - Crystallization and structure determination of compound 19

[0211] Compound 19 was prepated in 20 mM Tris pH 8, 150 mM KCI at a at a concentration of 10 mg / ml. Crystallization conditions were screened using a Crystal Gryphon liquid dispenser (Art Robbins Instruments) using a vapor diffusion method. Samples were disposed on MRC 96-Well Triple Drop Crystallization plates as sitting drops consisting of 0.2 pl of complex solution and 0.2 pl reservoir solution from different commercially available screens: JCSG Plus, PACT premier, SGI and BCS. The crystals grew in condition containing 2.4M Sodium malonate dibasic monohydrate pH 7.0.

[0212] No additional cryoprotection was neccessary and crystals were directly flash-frozen in liquid nitrogen. Crystal diffraction data was collected on a PROXIMA1 beamline at the SOLEIL synchrotron using EIGER-X 16M (Dectris Ltd.) detector (data statistics are given in Table 1). Structure was solved using molecular replacement (Phaser-MR) with insulin models (PDB 3140) as search models. After phasing, the initial structural model was manually rebuilt using Coot and iteratively refined using phenix. refine.

[0213] Table 1. Crystal data collection and refinement statistics analysis of modified insulin 19. P481445PC00 P481445PC00

[0214] Example 8 - Conclusion

[0215] We report the incorporation of non-natural NH-triazole, a close analogue of the histidine residue imidazole, into the protein structure. Biosynthetic incorporation of alkyne, followed by a copper- catalyzed reaction with hydrogen azide formed in situ from sodium azide in a slightly acidic aqueous buffer, provides a method for homogeneous protein preparation. In these cases, very fast kinetics were observed for copper-catalyzed reactions with protein substrates with recombinantly incorporated alkyne, suggesting acceleration by the protein surface. NH-triazoles can also be introduced into native proteins through conjugation of alkyne moieties and a subsequent copper- catalyzed reaction, or directly by conjugation with NH-triazole NHS esters, which allow the introduction of NH-triazole into any protein via various linkers. The high functional potential of NH- triazoles is demonstrated by the example of human insulin, whose thermal and chemical stability is significantly increased after the introduction of three NH-triazoles at Al-Gly, Bl-Phe and B29-Lys. The structure and biological activity of such insulin is retained even after slow heating to 95 °C, while the native form precipitates. Even after thermal treatment, the modified protein triggers the same signalling pathways as the native form, which ultimately leads to increased glucose uptake. In addition to the increased thermal stability, the described insulin is also chemically more stable towards insulin-degrading enzyme and has a higher affinity for metal ions than the wild-type. These results show the great potential of NH-triazole in the context of biomolecules. P481445PC00

[0216] Cited references i) Watson, J. L. et al. De novo design of protein structure and function with RFdiffusion. Nature 620, 1089-1100 (2023). ii) Chu, A. E., Lu, T. & Huang, P.o-S. Sparks of function by de novo protein design. Nat. Biotechnol. 42, 203-215 (2024). iii) Hartrampf, N. et al. Synthesis of proteins by automated flow chemistry. Science 368, 980-987 (2020). iv) Spicer, C. D. & Davis, B. G. Selective chemical protein modification. Nat. Commun. 5, 4740 (2014). v) Tamura, T. & Hamachi, I. Chemistry for Covalent Modification of Endogenous / Native Proteins: From Test Tubes to Complex Biological Systems. J. Am. Chem. Soc. 141, 2782-2799 (2019). vi) Liao, S.-M., Du, Q.-S., Meng, J.-Z., Pang, Z.-W. & Huang, R.-B. The multiple roles of histidine in protein interactions. Chem. Cent. J. 7, 44 (2013). vii) Chen, Y., Tao, K., Ji, W., Kumar, V. B., Rencus-Lazar, S. & Gazit, E. Histidine as a key modulator of molecular self-assembly: Peptide-based supramolecular materials inspired by biological systems. Mater. Today. 60, 106-127 (2022). viii) Barrett, A. J., Rawlings, N. D. & Woessner, J. F. Handbook of Proteolytic Enzymes. Academic Press, 3rd edition (2013). ix) Supuran, C. T. & Donald, W. A. Metalloenzymes: From Bench to Bedside. Academic Press, 1st edition, 2023. x) Wamhoff, H. 1,2,3-Triazoles and their Benzo Derivatives. Comprehensive Heterocyclic Chemistry. Vol. 5, pp. 669-732 (1984) xi) Hauang, D., Zhao, P. & Astruc, D. Catalysis by 1,2,3-triazole- and related transition-metal complexes. Coord. Chem. Rev. 272, 145-165 (2014). xii) Lauko, J., Kouwer, P. H. J. & Rowan, A. E. 1H-1, 2, 3, -Triazole: From Structure to Function and Catalysis. J. Heterocycl. Chem. 54, 1677-1699 (2016). P481445PC00 xiii) Jankovic, D., Virant, M. & Gazvoda, M. Copper-Catalyzed Azide-Alkyne Cycloaddition of Hydrazoic Acid Formed In Situ from Sodium Azide Affords 4-Monosubstituted-l,2,3-Triazoles. J. Org. Chem. 87, 4018-4028 (2022) and see references therein for synthetic protocols for NH-triazole incorporation. xiv) Isenegger, P. G. & Davis, B. G. Concepts of Catalysis in Site-Selective Protein Modifications. J. Am. Chem. Soc. 141, 8005-8013 (2019). xv) Vinogradova, E. V., Zhang, C., Spokoyny, A. M., Pentelute, B. L. & Buchwald, S. L. Organometallic palladium reagents for cysteine bioconjugation. Nature 526, 687-691 (2015). xvi) McDonald, J. R.; Rabalais, J. W. & McGlynn, S. P. Electronic Spectra of the Azide Ion, Hydrazoic Acid, and Azido Molecules. J. Chem. Phys. 52, 1332-1340 (1970). xvii) Wang, L. & Schultz, P. G. Expanding the Genetic Code. Angew. Chem. Int. Ed. 44, 34-66 (2005). xviii) Young, T. S., Ahmad, I., Yin, J. A. & Schultz, P. G. An Enhanced System for Unnatural Amino Acid Mutagenesis in E. coli. J. Mol. Biol. 395, 361-374 (2010). xviv) Deiters, A., Cropp, T. A., Mukherji, M., Chin, J. W., Anderson, J. C. & Schultz, P. G. Adding Amino Acids with Novel Reactivity to the Genetic Code of Saccharomyces Cerevisiae. J. Am. Chem. Soc. 125, 11782-11783 (2003). xx) Suzuki, S., Tatsuguchi, A., Matsumoto, E., Kawazoe, M., Kaminishi, T., Shirouzu, M., Muto, Y., Takemoto, C. & Yokoyama, S. Structural Characterization of the Ribosome Maturation Protein, RimM. J. Bacteriol. 189, 6397-6406 (2007). xxi) Dumas, P., Bergdoll, M., Cagnon, C. & Masson, J. M. Crystal structure and site - directed mutagenesis of a bleomycin resistance protein and their significance for drug sequestering. EMBO J. 13, 2483-2492 (1994). xxii) Shen, J., Griffiths, P. T., Campbell, S. J., Utinger, B., Kalberer, M. & Paulson, S. E. Ascorbate oxidation by iron, copper and reactive oxygen species: review, model development, and derivation of key rate constants. Sci. Rep. 11, 7417 (2021). P481445PC00 xxiii) Li, S., Cai, H., He, J., Chen, H., Lam, S., Cai, T., Zhu, Z., Bark, S. J. & Cai, C. Extent of the Oxidative Side Reactions to Peptides and Proteins During the CuAAC Reaction. Bioconjugate Chem. Tl, 2315-2322 (2016). xxiv) Mathieu, C., Martens, P.-J. & Vangoitsenhoven, R. One hundred years of insulin therapy. Nat. Rev. Endocrinol. 17, 715-725 (2021). xxv) Green, N. M. Avidin and streptavidin. Methods Enzymol. 184, 51-67 (1990). xxvi) Plosker, G. L. & Keam, S. J. Trastuzumab: a review of its use in the management of HER2- positive metastatic and early-stage breast cancer. Drugs 66, 449-475 (2006). xxvii) Zuma, L. K., Gasa, N. L., Makhoba, X. H. & Pooe, O. J. Protein PEGylation: Navigating Recombinant Protein Stability, Aggregation, and Bioactivity. Biomed. Res. Int. 8929715 (2022). xxviii) Palm, T., Esfandiary, R. & Gandhi, R. The effect of PEGylation on the stability of small therapeutic proteins. Pharm. Dev. Technol. 16, 441-448 (2011). xxix) Sen, S., Ali, R., Onkar, A., Verma, S., Ahmad, Q. T., Bhadauriya, P., Sinha, P., Nair, N. N., Ganesh, S. & Verma, S. Synthesis of a highly thermostable insulin by phenylalanine conjugation at B29 Lysine. Commun. Chem. 7, 161 (2024). xxx) Leader, B., Baca, Q. J. & Golan, D. E. Protein therapeutics: a summary and pharmacological classification. Nat. Rev. Drug Discov. 7, 21-39 (2008). xxxi) Akbarian, M. & Chen, S.-H. Instability Challenges and Stabilization Strategies of Pharmaceutical Proteins. Pharmaceutics 14, 2533 (2022). xxxii) Basle, E., Joubert, N. & Pucheault, M. Protein Chemical Modification on Endogenous Amino Acids. Chem. Biol. 17, 213-227 (2010). xxxiii) Fischer, N. H., Oliveir, M. T. & Diness, F. Chemical modification of proteins - challenges and trends at the start of the 2020s, Biomater. Sci. 11, 719-748 (2023). xxxiv) Johnson, I. S. Human insulin from recombinant DNA technology. Science 219, 632-637 (1983). xxxv) Banting, F. G., Best, C. H., Collip, J. B., Campbell, W. R. & Fletcher, A. A. Pancreatic Extracts in the Treatment of Diabetes Mellitus. Can. Med. Assoc J. 12, 141-146 (1922). P481445PC00 xxxvi) Zaykov, A. N., Mayer, J. P. & DiMarchi, R. D. Pursuit of a perfect insulin. Nat. Rev. Drug Discov. 15, 425-439 (2016). xxxvii) Das, A., Shah, M. & Saraogi, I. Molecular Aspects of Insulin Aggregation and Various Therapeutic Interventions. ACS Bio. Med. Chem. Au 2, 205-221 (2022). xxxviii) Hubalek, F. et. al. Molecular engineering of safe and efficacious oral basal insulin. Nat. Commun. 11, 3746 (2020). xxxix) Marco-Dufort, B. et al. Thermal stabilization of diverse biologies using reversible hydrogels. Sci. Adv. 8, eabo0502 (2022). xl) Webber, M. J., Appel, E. A., Vinciguerra, B., Cortinas, A. B., Thapa, L. S., Jhunjhunwala, S., Isaacs, L., Langer, R. & Anderson, D. G. Supramolecular PEGylation of Biopharmaceuticals. Proc. Natl. Acad. Sci. 113, 14189-14194 (2016). xli) Xiong, X., et al. Novel Four-Disulfide Insulin Analog with High Aggregation Stability and Potency. Chem. Sci. 11, 195-200 (2020). xlii) Steiner, D. F., Cunningham, D., Spigelman, L. & Aten, B. Insulin biosynthesis: evidence for a precursor. Science 157, 697-700 (1967). xliii) Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Bing, X. & Markussen, J. X-ray analysis of the single chain B29-A1 peptidelinked insulin molecule. A completely inactive analogue. J. Mol. Biol. 220, 425-433 (1991). xliv) Hua, Q. X., Shoelson, S. E., Kochoyan, M., & Weiss, M. A. Receptor binding redefined by a structural switch in a mutant human insulin. Nature 354, 238-241 (1991). xlv) Bohr, F. et al. Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies. Commun. Biol. 6, 178 (2023). xlvi) Brange, J. & Langkjar, L. Insulin Structure and Stability (pp 315-350) in Stability and Characterization of Protein and Peptide Drugs. (Eds. Pearlman, R. & Wang, Y. J.), 3rd Edition, Springer (1993). xlvii) Agalave, S. G., Maujan, S. R. & Pore, V. S. ClickChemistry: 1,2,3-Triazoles as Pharmacophores.

[0217] Chem. Asian J. 6, 2696-2718 (2011). P481445PC00 xlviii) Bozorov, K., Zhao, J. & Aisa, H. A. 1,2,3-Triazole-containing hybrids as leads in medicinal chemistry: A recent overview. Bioorg. Med. Chem. 27, 3511-3531 (2019). xlviv) Angelo, N. G. & Arora, P. S. Nonpeptidic Foldamers from Amino Acids: Synthesis and Characterization of 1,3-Substituted Triazole Oligomers. J. Am. Chem. Soc. 127, 17134-17135 (2005).

[0218] I) Bourne, Y., Kolb, H. C., Radic, Z., Sharpless, K. B., Taylor, P. & Marchot, P. Freeze-frame inhibitor captures acetylcholinesterasein a unique conformation. Proc. Natl. Acad. Sci. USA 101, 1449 (2004).

[0219] II) Agouram, N., Hadrami, E. M. E. & Bentama, A. 1,2,3-Triazoles as Biomimetics in Peptide Science. Molecules 26, 2937 (2021).

[0220] Hi) Li, Y. & Flood, A. H. Pure C-H Hydrogen Bonding to Chloride Ions: A Preorganized and Rigid Macrocyclic Receptor. Angew. Chem. Int. Ed. 47, 2649 (2008)..

[0221] Illi) Wu, C.-F., Li, Z.-M., Xu, X.-N., Zhao, Z.-X., Zhao, X., Wang, R.-X. & Li, Z.-T. Folding-Induced Folding: The Assembly of Aromatic Amide and 1,2,3-Triazole Hybrid Helices. Chem. Eur. J. 20, 1418- 1426 (2014). liv) Juwarker, H., Lenhardt J. M., Pham, D. M. & Craig, S. L. 1,2,3-Triazole CH—CI Contacts Guide Anion Binding and Concomitant Folding in 1,4-Dia ryl Triazole Oligomers. Angew. Chem. Int. Ed. 47, 3740-3743 (2008).

[0222] Iv) Meudtner, R. M. & Hecht, S. Helicity Inversion in Responsive Foldamers Induced by Achiral Halide ion Guests. Angew. Chem. Int. Ed. 47, 4926-4930 (2008).

[0223] Ivi) Glidden, M. D. et al. An Ultra-Stable Single-Chain Insulin Analog Resists Thermal Inactivation and Exhibits Biological Signaling Duration Equivalent to the Native Protein. J. Biol. Chem. 293, 47-68 (2018).

[0224] Ivii) Tundo, G. R., Grasso, G., Persico, M., Tkachuk, O., Bellia, F., Bocedi, A., Marini, S., Parravano, M., Graziani, G., Fattorusso, C. & Sbardella, D. The Insulin-Degrading Enzyme from Structure to Allosteric Modulation: New Perspectives for Drug Design. Biomolecules 13, 1492 (2023).

Claims

P481445PC00Claims1. Polypeptide comprising a NH-l,2,3-triazole.

2. The polypeptide according to claim 1, wherein the NH-l,2,3-triazole is attached to the remaining part of the polypeptide at the carbon in position 4 or 5.

3. The polypeptide according to claim 1 or 2, wherein the NH-l,2,3-triazole has the structure and attachment to the polypeptide R as in any one of Formulae 1-1 to 1-V or a mixture thereof :Formulae 1-1 to 1-V4. The polypeptide according to any of the preceding claims, wherein the NH-l,2,3-triazole has the structure and attachment to the polypeptide R as in any one of Formulae 1-1 to 1- III or a mixture thereof.

5. The polypeptide according to claim 1 or 2, wherein the NH-l,2,3-triazole has the structure and attachment to the polypeptide as in any one of Formulae 2-1 to 2-V or a mixture thereofFormulae 2-1 to 2-V6. The polypeptide according to any one of claims 1-2 and 5, wherein the NH-l,2,3-triazole has the structure and attachment to the polypeptide R as in any one of Formulae 2-1 to 2- III or a mixture thereof.P481445PC007. The polypeptide according to any of the claims 1-2 and 5-6, wherein the NH-l,2,3-triazole is part of the non-proteogenic alpha amino acid residue of any one of Formula 3-1 to 3-V or a mixture thereof :Formulae 3-I to 3-V8. The polypeptide according to any one of claims 5-7, wherein the NH-l,2,3-triazole is part of the non-proteogenic alpha amino acid residue of any one of Formula 3-I to 3-III or a mixture thereof.

9. The polypeptide according to any of the preceding claims, wherein the NH-l,2,3-triazole is part of the non-proteogenic alpha amino acid residue :NH-l,2,3-triazole - LI - CHNH2COOH, wherein LI is a linker.

10. The polypeptide according to claim 9, wherein LI is an optionally substituted Ci-25-alkyl.

11. The polypeptide according to claim 9 or 10, wherein LI is an optionally substituted linear Ci-25-alkyl.

12. The polypeptide according to any one of claims 9-11, wherein LI is a Ci-25-alkyl.

13. The polypeptide according to any one of claims 9-12, wherein LI is a linear Ci-25-alkyl.

14. The polypeptide according to any one of claims 9-13, wherein LI is selected from the group consisting of methyl, ethyl, propyl and n-butyl.

15. The polypeptide according to claim 9, wherein LI comprises a polyethylene (PEG) moiety.

16. The polypeptide according to claim 15, wherein the PEG moiety is polydisperse.39P481445PC0017. The polypeptide according to claim 15, wherein the PEG moiety is monodispersed.

18. The polypeptide according to any one of claims 15-17, wherein the PEG moiety has a molecular weight of less than 4000 kDa, less than 2000 kDa or less than 1000 kDa.

19. The polypeptide according to any one of claims 15-18, wherein the PEG moiety has the structure -(CH2-0-CH2)n- wherein n is from 1 to 50, from 1 to 25 or from 2 to 15.

20. The polypeptide according to claim 19, wherein n is 6, 8 or 10.

21. The polypeptide according to any one of the preceding claims, wherein the NH-1,2,3- triazole is attached to the epsilon-amino group of a lysine or arginine residue.

22. The polypeptide according to any one of the preceding claims, wherein the NH-1,2,3- triazole is attached to the epsilon-amino group of a lysine residue.

23. The polypeptide according to any one of the preceding claims, wherein the NH-1,2,3- triazole is attached to the N-terminal amino group of the polypeptide.

24. The polypeptide according to any one of the preceding claims, wherein the NH-1,2,3- triazole has replaced an imidazole of a histidine residue of the polypeptide in its naturally occurring form.

25. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises more than one NH-l,2,3-triazole.

26. The polypeptide according to any one of the preceding claims, which is a derivative of human insulin, desB30-human insulin, or a human insulin analogue.

27. The polypeptide according to any one of the preceding claims, which is a derivative of human insulin, desB30-human insulin, or a human insulin analogue comprising a NH-1,2,3- triazole attached to B29-Lys.

28. The polypeptide according to any one of claims 25-27, which is a derivative of human insulin, desB30-human insulin or a human insulin analogues comprising a NH-l,2,3-triazole attached to Bl-Phe.

29. The polypeptide according to any one of claims 25-28, which is a derivative of human insulin, desB30-human insulin or a human insulin analogues comprising a NH-l,2,3-triazole attached to Al-Gly.40P481445PC0030. The polypeptide according to any one of claims 25-29, which is human insulin or desB30 human insulin having three NH-l,2,3-triazoles attached to Al-Gly, Bl-Phe and B29-Lys, respectively.

31. The polypeptide according to any one of claims 25-30, which is human insulin being or DesB30-human insulin derivatized with a structure comprising a NH-l,2,3-triazole at B29- Lys, Bl-Phe or Al-Gly.

32. The polypeptide according to any one of claims 25-31, which is human insulin or DesB30- human insulin being derivatized with a structure comprising a NH-l,2,3-triazole at the position : B29-Lys and Bl-Phe, B29-Lys and Al-Gly or Bl-Phe and Al-Gly.

33. The polypeptide according to any one of claims 1-25, which is a NH-1, 2, 3, -triazole functionalized variant of human insulin, ribosome maturation factor (RIMM), bleomycin resistance protein (BRP), green fluorescent protein (GFP), superfolder GFP (sfGFP) or avidin.

34. The polypeptide according to any one of claims 1-25, which is a NH-1, 2, 3, -triazole functionalized variant of an antibody.

35. The polypeptide according to claim 34, wherein the antibody is trastuzumab.

36. The polypeptide according to any one of claims 1-24, which is a GLP-1 agonist.

37. The polypeptide according to claim 36, which is a GLP-1 peptide derivative.

38. The polypeptide according to claim 36 or 37, which is a NH-1, 2, 3, -triazole functionalized variant of dulaglutide, exenatide, semaglutide, liraglutide or lixisenatide.

39. The polypeptide according to any one of the preceding claims, which is formed by introducing an alkyne and performing a copper catalyzed cycloaddition of hydrogen azide to the alkyne.

40. The polypeptide according to any one of the preceding claims, which is formed by bioconjugation of one or more basic protein sites by alkyne-functionalized N- hydroxysuccinimide (NHS) ester followed by copper catalyzed cycloaddition of hydrogen azide to the alkyne.

41. The polypeptide according to any one of claims 1-39, where the introduction of an alkyne is made by genetic code expansion technology.P481445PC0042. The polypeptide according to any one of claims 1-39, wherein basic residues of the protein is reacted with NH-l,2,3-triazole functionalized NHS ester.

43. Method for preparing the polypeptide as defined in any one of claims 1-41, comprising the steps : a) providing the protein to be NH-l,2,3-triazole functionalized, b) introducing at least one alkyne into the protein to form an alkyne-functionalized protein, c) reacting the alkyne functionalized protein with hydrogen azide in the presence of a copper catalyst, and c) isolating said polypeptide.

44. The method according to claim 43, wherein in step b) the at least one alkyne is introduced by bioconjugation of one or more basic protein sites by alkyne-functionalized NHS ester.

45. The method according to claim 43, wherein in step b) the at least one alkyne is introduced by genetic code expansion technology.

46. The method according to any one of claims 43-45, wherein in step c) the copper catalyst is introduced as a Cu(ll) or Cu(l) salt with addition of an ascorbate salt.

47. The method according to claim 46, wherein in step c), a CuAAC-accelerating ligand, e.g. THPTA, is added.

48. The method according to any one of claims 43-47, wherein in step c) the copper catalyst is CuSO4 in combination with sodium ascorbate and water-soluble CuAAC-accelerating ligand THPTA.

49. The method according to any one of claims 43-48, wherein the reaction is conducted in a buffer at a pH in the range from 1.0 to 8.0, and at a temperature in the range from 10 °C to 90 °C.

50. The method according to any one of claims 43-49, wherein the reaction is conducted in a buffer at a pH in the range from 4.0 to 8.0, and at a temperature in the range from 10 °C to 90 °C.

51. The method according to any one of claims 43-50, wherein the reaction is conducted at a pH in the range from 4.0 to 6.0.P481445PC0052. The method according to any one of claims 43-51, wherein the reaction is conducted at a temperature in the range from 10 °C to 35 °C.

53. The method according to any one of claims 43-52, wherein the reaction is conducted at a pH of about 5 and at room temperature.

54. Method for preparing a polypeptide as defined in any one of claims 1-38 and 42, comprising the steps : g) providing the protein to be NH-l,2,3-triazole functionalized, h) providing a NH-l,2,3-triazole functionalized NHS ester, c) reacting the protein with NH-l,2,3-triazole functionalized NHS ester, and d) isolating said polypeptide.

55. The method according to claim 54, wherein in step b) the NH-1, 2, 3, -triazole functionalizedNHS ester has the structure:NHS-L2-NH-triazole, wherein NHS is N-hydroxy-succinimide and the linker L2 is selected from the group consisting of Ci-25-alkyl, linear Ci-25-alkyl, optionally substituted Ci-25-alkyl, optionally substituted linear Ci-25-alkyl, methyl, ethyl, propyl, n-butyl, and polyethylene (PEG) moiety.

56. The method according to claim 54 or 55, wherein in step b) the NH-l,2,3-triazole functionalized NHS ester is selected from compounds 23-262,5-dioxopyrrolidin-l-yl lH-l,2,3-triazole-5-carboxylate (23), 2,5-dioxopyrrolidin-l-yl 3- (lH-l,2,3-triazol-5-yl)propanoate (24), 2,5-dioxopyrrolidin-l-yl 3-((lH-l,2,3-triazol-5-43P481445PC00 yl)methoxy)propanoate (25), 2,5-dioxopyrrolidin-l-yl 3-(2-(2-((lH-l,2,3-triazol-5- yl)methoxy)ethoxy)ethoxy)propa noate (26).

57. The method according to any one of claims 54-56, wherein in step b) the NH-l,2,3-triazole functionalized NHS ester is prepared by reaction of NH-l,2,3-triazole functionalized carboxylic acid with N,N'-disuccinimidyl carbonate (DSC).

58. The method according to any one of claims 54-57, wherein the reaction of step c) is performed at a temperature from 10 °C to 70 °C, at a pH from 7.1 to 11.0.

59. The method according to any one of claims 54-58, wherein the reaction of step c) is performed at a temperature from 10 °C to 70 °C, at a pH from 7.5 to 9.5.

60. The method according to any one of claims 54-59, wherein the reaction of step c) is performed at a temperature from 60 °C to 70 °C, at a pH from 7.5 to 9.5.

61. The method according to any one of claims 54-60, wherein the reaction in steps c) is performed at a temperature of about 65 °C and a pH of about 8.5.

62. The method according to any one of claims 54-61, wherein in step c) the reaction is performed in a buffer.

63. The method according to any one of claims 54-62, wherein in step c) the reaction is performed in a buffer which is e.g., phosphate buffered saline (PBS), TRIS, phosphate buffers, bicarbonate buffers, protein buffers and Good's buffers.

64. Method for preparing a polypeptide being a stabilized variant of a protein having a certain biological function, which method comprises introducing at least one lH-l,2,3-triazole moiety into said protein, thereby providing a polypeptide having increased stability and substantially the same biological function.

65. Method for purifying a polypeptide which comprises at least one lH-l,2,3-triazole moiety, wherein said method comprises metal affinity chromatography.

66. The method according to item 65, wherein the metal affinity chromatography is Cu2+affinity chromatography, Ni2+affinity chromatography, Co2+affinity chromatography, Zn2+, affinity chromatography or Fe3+ / Fe2+affinity chromatography.44P481445PC0067. The method according to claim 65 or 66, wherein the metal affinity chromatography is Cu2+affinity chromatography68. The method according to any one of claims 65-67, wherein a solution comprising said polypeptide comprising at least one lH-l,2,3-triazole moiety is applied to a metal affinity chromatography material, and wherein said solution has a pH of less than 6.0.

69. The method according to any one of claims 65-68, wherein said metal affinity chromatography comprises two-step elution where the first elution is by first eluent being a buffer having a pH from about 3.0 to about 5.0 and where the first eluent does not contain 1,2,3-triazole, and where the second elution is by a second eluent being a buffer having a pH from about 3.0 to about 5.0 and where the second eluent does comprise 1,2,3-triazole.

70. The method according to claim 69, wherein the buffer in the first eluent and in the second eluent are both an acetate buffer.

71. The method according to any one of claims 64-70, wherein said polypeptide is as defined in any one of claims 1-42.

72. Pharmaceutical formulation comprising the polypeptide as defined in any one of claims 1-42 and at least one pharmaceutically acceptable excipient.

73. The pharmaceutical formulation according to claim 72, which is an aqueous formulation.

Images