Multi-equilibria system enables precise carboxylic acid engineering in proteins

Abstract

The present invention discloses a method for precision engineering of protein bioconjugates by harnessing complex multi-equilibria systems. The method allows for exclusive control over reaction pathways, achieving highly selective outcomes, even with complex substrates such as proteins. By manipulating proteinogenic functionalities, the invention enables pathway selectivity, chemoselectivity, and site selectivity, leading to single-site modifications in proteins. The versatility of the present invention is demonstrated through diverse protein examples, including insulin, RNase A, ubiquitin, cytochrome C, myoglobin, α-lactalbumin, and serum albumins, showcasing its broad applicability in protein engineering. Additionally, the method facilitates the synthesis of antibody-fluorophore conjugates (AFC) and antibody-drug conjugates (ADC), providing promising prospects for therapeutic applications.

Description

[0001] MULTI-EQUILIBRIA SYSTEM ENABLES PRECISE CARBOXYLIC ACID ENGINEERING IN PROTEINS

[0002] FIELD OF INVENTION

[0003] The present invention relates to the field of chemical biology and bioconjugation chemistry. More particularly, it pertains to a multi -equilibria reaction platform for site- selective and chemoselective modification of biomolecules, including proteins, enzymes, and monoclonal antibodies.

[0004] BACKGROUND OF THE INVENTION

[0005] The principles of organic chemistry were developed to meet the needs of the chemical industry. As a result, there was a focus on creating clean reactions, high yields, and the most efficient pathways to products. However, this focus led to a preference for irreversible transformations, creating a divide between synthetic and biological chemistry. Biological systems have multiple chemical pathways that allow for adaptation, evolution, and switching between pathways. Synthetic biology has been able to harness these capabilities to engineer new functions in various biological components. Reversibility, whether dynamic or triggered, is crucial for these systems to adapt and evolve. The gap between the evolution of chemical and biological methods provides an excellent opportunity for discovery.

[0006] The exploration of multi-equilibria systems in organic chemistry has mainly been limited to combinatorial pathways. The complexity of these systems has made it difficult to obtain chemically pure products and has hindered their application to biomolecules, such as proteins, which can interact with multiple components due to their numerous functional groups. This added complexity has made it even more challenging to work with these systems in organic chemistry in conjunction with proteins.

[0007] However, precise engineering of proteins is essential to meet various technological needs at the interface of biology and medicine, requiring a chemical method to deliver a combination of reactivity and selectivity attributes. Initially, protein bioconjugation methods focused on irreversible reactions along the shortest pathways. Over the past few decades, efforts have centered on targeting the reactivity hotspots offered by proteins.

[0008] In recent years, it is demonstrated that the initial potential of reversible pathways to disintegrate the selectivity attributes, priming chemical methods to offer residue and protein specificity beyond the reactivity order defined by the proteins.

[0009] The present invention demonstrates a multi-equilibria system that provides access to a wide range of intermediates capable of selecting and evolving a precisely labelled bioconjugate. These intermediates can exclusively initiate three distinct multicomponent reactions without interference from other pathways. This unique reactivity and selectivity landscape allows for the chemoselective and site- selective labelling of carboxylic acid in proteins, enzymes, and a monoclonal antibody (mAb). Despite carboxylate ranking low in the nucleophilicity order, the methods result in the exclusive labelling of a single site and enable control over modularity. This, in turn, allows for the targeting of mutually exclusive residues to deliver single-site bioconjugation. Contrary to conventional wisdom, the complexity of the protein surface simplifies the outcome of a multi-equilibria system, allowing for a pathway- selective four-component reaction (4CR) between the protein's carboxylate, hydroxylamine hydrochloride, aldehyde, and isonitrile producing a four-component product (4CP).

[0010] The selection of substrates facilitates the disruption of 4CR-equilibria to produce a two-component product (2CP). This pathway challenges core chemistry concepts wherein an intramolecular reaction, favoured due to proximity effects, is entirely intercepted and overridden by a competing intermolecular reaction. In one embodiment of the present invention, a protein-based carboxylate reacts with hydroxylamine and isonitrile in a three-component reaction (3CR) to produce a 2CP. The hydroxylamine component substituted by other nucleophiles, including but not limited to primary amines, hydrazines, and azides. This substitution enables the 3CR- 2CP to create distinct bond architecture. Additionally, it provides synthetic flexibility to install azide, alkyne, fluorophore, and NMR probes. The present method also extends seamlessly for dual-probe installation in a protein, allowing the installation of a FRET acceptor and donor at one or two sites at a well-defined distance. The selectivity of the overall platform (4CR-4CP, 4CR-2CP, and 3CR-2CP) is demonstrated in more than thirty-six examples using structurally diverse proteins. Furthermore, it is extended to pathway-selective, chemoselective, site-selective, and modular insulin labelling. Importantly, it does not impact its binding with the insulin receptor or the downstream signalling pathway. Finally, the present method is employed for synthesizing homogeneous antib ody-fluorophore conjugates (AFC) and antibody-drug conjugate (ADCs). The AFC offers antigen-specific cellular imaging, while the ADCs exhibit potent and selective anti-proliferative activity against HER2 -positive SKBR-3 breast cancer cells.

[0011] OBJECTIVES OF THE INVENTION

[0012] The main objective of the present invention relates to a method for the precision engineering of biomolecule bioconjugation utilizing multi-equilibria systems.

[0013] Another objective of the present invention is to facilitate the precise labelling of bioconjugates by enabling multiple reaction pathways.

[0014] Yet another objective of the present invention is to provide the unique reactivity and selectivity landscapes, achieving chemoselective and site-selective labeling of carboxylic acids in proteins, enzymes, and monoclonal antibodies (mAbs).

[0015] SUMMARY OF THE INVENTION

[0016] The present invention relates to a novel method for protein bioconjugation leveraging multi -equilibria systems that enable selective and site-specific labelling of carboxylate residues in proteins, enzymes, and monoclonal antibodies (mAbs). The present method allows the precise installation of various probes, including fluorophores and nuclear magnetic resonance (NMR) tags, and the synthesis of homogeneous antib ody-fluorophore conjugates (AFCs) and antibody-drug conjugates (ADCs). The present invention showcases three distinct multicomponent reactions (MCRs): a. Four-component reaction leading to a four-component product (4CR-4CP). b. Four-component reaction leading to a two-component product (4CR-2CP). c. Three-component reaction leading to a two-component product (3CR-2CP).

[0017] These pathways provide unique reactivity and selectivity landscapes, enabling chemoselective and site-selective labelling of carboxylates, despite their low nucleophilicity. This platform is validated with over thirty-six examples using diverse proteins and extended to the modular labelling of insulin without affecting its receptor binding or signalling pathways. Additionally, the present developed methods for synthesizing homogeneous AFCs and ADCs with high selectivity and functionality.

[0018] In one aspect of the present invention, a method for site-selective bioconjugation of proteins via a multi-equilibria disruption mechanism utilizing a four-component reaction-two-component product (4CR-2CP) pathway, the method comprises the steps of:

[0019] (a) providing a protein comprising at least one proteinogenic carboxylate side chain;

[0020] (b) reacting said protein with:

[0021] (i) an aldehyde compound of Formula (3);

[0022] (ii) an isonitrile compound of Formula (4), wherein said isonitrile is tert-butyl isonitrile (4a); and

[0023] (iii) a nucleophile selected from hydroxylamines and hydrazines of Formula (2);

[0024] (c) selecting said aldehyde from a plurality of aldehydes, wherein n-butyraldehyde (3b), in combination with tert-butyl isonitrile (4a) and (9-benzylhydroxylamine hydrochloride (2c), achieves exclusive 4CR-2CP pathway selectivity with single-site modification at aspartate residue DI 19 of lysozyme C;

[0025] (d) optionally using aromatic aldehydes including 2-hydroxybenzaldehyde (3e), benzaldehyde (3f), and 3,4-dihydroxybenzaldehyde (3g), which also result in site- selective labeling via the 4CR-2CP pathway; (e) occuring bioconjugation under reaction conditions effective to selectively modify one or more carboxylate sites in said protein, and

[0026] (f) forming a site-selectively labeled protein conjugate via either a 4CR-2CP or a three-component reaction-two-component product (3CR-2CP) mechanism.

[0027] Another aspect of the present invention, the aldehyde is selected from aliphatic aldehydes including n-butyraldehyde (3b), aromatic aldehydes including 2- hy dr oxybenzaldehyde (3e), benzaldehyde (3f), 3,4-dihydroxybenzaldehyde (3g), and heteroaromatic aldehydes.

[0028] Another aspect of the present invention, the protein is selected from the group consisting of RNase A, ubiquitin, cytochrome C, myoglobin, a-lactalbumin, bovine serum albumin (BSA), human serum albumin (HSA), and antibodies including trastuzumab.

[0029] Yet another aspect of the present invention, site-selective carboxylate modification occurs at a residue selected from DI 19 or D18 of lysozyme C, D121 of RNase A, D52 of ubiquitin, D2 of cytochrome C, D109 of myoglobin, El l of a-lactalbumin, D258 or D268 of BSA, or E252 of HSA.

[0030] Yet another aspect of the present invention, the reaction is carried out at a pH between 6.0 and 7.0 and a temperature between 20°C and 30°C.

[0031] Yet another aspect of the present invention, the protein is a model peptide containing a single glutamate residue, and only the glutamate residue is modified.

[0032] Yet another aspect of the present invention, the protein is insulin, and the modification occurs selectively at residue El 7, yielding a mono-labeled insulin bioconjugate.

[0033] Yet another aspect of the present invention, the aldehyde is formaldehyde (3a), the isonitrile is tert-butyl isonitrile (4a), the nucleophile is hydrazine hydrochloride (2k), and the reaction is performed in HEPES buffer at pH 7.0, 20% acetonitrile, at 25°C using 200 equivalents of hydrazine. Yet another aspect of the present invention, the reaction is conducted at a protein concentration of approximately 73 pM, with aldehyde and isonitrile at approximately 73 mM, for duration of about 8 hours, resulting in at least 96% conversion.

[0034] Yet another aspect of the present invention, a method for dual-fluorophore labeling of proteins for Forster Resonance Energy Transfer (FRET) assays, comprising:

[0035] (a) performing the 3CR-2CP bioconjugation method as claimed in claim 1 to attach a first fluorophore at a designated carboxylate residue; and

[0036] (b) performing a thiolyne reaction to attach a second fluorophore at a cysteine or other thiol-containing residue.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The various implementations disclosed herein are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings, in which like reference numerals may refer to similar elements.

[0039] Figure 1: illustrates the multi-equilibria systems involving proteins open new possibilities by unravelling complexity to enable a single irreversible pathway. Achieving pathway selectivity, along with chemoselectivity and site-selectivity, provides methods for the precise engineering of proteins. Representative pathways involve proteins, isonitriles, aldehydes, and external amines, including: Path I, where proteinogenic amines form imines; Path II, involving tryptophan; Path III, possibly involving tyrosine; and Paths IV and V, which involve carboxylates through distinct routes. "CR" (component reaction) refers to the number of reagents participating in a pathway, while "CP" (component product) refers to the number of reagents present in the bioconjugate, in accordance with the present invention;

[0040] Figure 2: illustrates the Four-component based multi-equilibria systems. The reaction of protein (la) with external amine (2a), aldehyde (3), and isonitrile (4a). a. Formaldehyde (3a) engages Lys (KI), Glu (E7, E35), and Asp (DI 19) in 4CR-3CP along with Asp (DI 8, D48, D52, D101, DI 19) in 4CR-4CP. Simultaneous 4CR-4CP and 4CR-3CP are also observed, b. For aliphatic aldehyde (3b), the Lys (KI) from lysozyme C (la) participates in the 4CR-3CP (10%) and Asp (D66) results in a 4CR- 4CP (4%). C. The effect of aldehydes (3a-3f) on the selectivity attributes, d. The effect of isonitriles (4a-4d) on the selectivity attributes, e. Pathway-selective, chemoselective, and site-selective 4CR-4CP with structurally diverse proteins (laid), in accordance with the present invention;

[0041] Figure 3: illustrates the disruption of 4CR-4CP to 4CR-2CP. a. multi-equilibria system with la, 2c, 3a, and 4a. b. re-routing 4CR-4CP to 4CR-2CP requires interception of a facile intramolecular nucleophilic addition by an intermolecular reaction, a first example in this category, c. The effect of aldehydes (3b-3f) on the selectivity attributes, d. The effect of isonitriles (4a-4d) on the selectivity attributes, in accordance with the present invention;

[0042] Figure 4: depicts the 4CR-2CP offers aldehyde-enabled modularity with structurally diverse proteins, a. The reaction of proteins (la-lh) with other components (2c, 3b, and 4a). b, The reaction of protein (la) with other components (2c, 3g, and 4a). Pathway selectivity remains conserved along with chemoselectivity and site-selectivity, in accordance with the present invention;

[0043] Figure 5: illustrates the three-component based multi-equilibria systems, mono- and dual-probe engineering, and biophysical investigation, a. Mechanistic possibilities with 3CR-2CP. Out of multiple options, can carboxylate-isonitrile adduct react exclusively with the hydroxylamine to render 3CR-2CP, b. The reaction of proteins (la-lh) with other components (2c and 4a). 3CR-2CP enables access to a new targetome while retaining all the selectivity attributes, c. Versatile functional group engineering - installation of chemically orthogonal handles (52-54), probes (55, 56), and bond architectures (57, 58), in accordance with the present invention.

[0044] Figure 6: depicts the dual-probe engineering, and biophysical investigation, a. Step 1 - BSA (1g) with hydroxylamine hydrochloride (2e) and isonitrile (4a) renders 3CR-2CP selective E45-alkyne bioconjugate (59). Step 2 - Thiol-yne reaction with 59 and DBCO-Cy5-dye (60) results in C34-Cy5, E45-alkyne dual-labeled bioconjugate (61). Step 3 E45-alkyne bioconjugate (59) subjected to chemically orthogonal cycloaddition with azide functionalized Cy3 (62) renders 63. Steps 2 and 3 performed sequentially with 59 for synthesis of C34-Cy5, E45-Cy3 dual-labeled bioconjugate (64). b. Normalized spectra of E45-Cy3 (63, donor) emission and C34- Cy5 (61, acceptor) absorption for 64. c. Normalized emission spectra of the E45-Cy3 labeled 63 and C34-Cy5, E45-Cy3 dual-labeled 64 (kex = 550 nm). d. Fluorescence lifetime decay transients of E45-Cy3 labeled 63 (red) and C34-Cy5, E45-Cy3 duallabeled 64 (green) (kex = 560 nm; kern = 570 nm). e. Step 1 - BSA (1g) with hydroxylamine hydrochloride (2f) and 4a renders C-ter-alkyne / azide dual probe bioconjugate (65). Step 2 - Metal-free click reaction with DBCO-Cy5-dye (60) results in C-ter-alkyne / Cy5 dual probe bioconjugate (66). Step 3 - Bioconjugate 65 subjected to a cycloaddition with azide functionalized Cy3 (62) renders 67. Steps 2 and 3 performed sequentially with 65 for synthesis of C-ter-Cy3 / Cy5 dual-labeled bioconjugate (68). f. Normalized fluorescence emission spectra of 67 and 68 (kex = 550 nm). g. Fluorescence lifetime decay spectra of the 67 (red) and 68 (green) (kex = 560 nm; kern = 570 nm). Terminal alkyne in 59 was examined for CuAAC with Cy3- azide (62; bioconjugate 63). Next, the optimal conditions used with C34-Cy5ZE45- alkyne labeled BSA (61) to result in C34-Cy5ZE45-Cy3 dual-probe-labeled BSA (64). The structure of 64 remains unperturbed with respect to the native BSA, in accordance with the present invention;

[0045] Figure 7: illustrates the precision engineering of carboxylate in insulin and trastuzumab (AFC and ADC). Insulin modification with a. 3CR-2CP pathway (li, 2c, and 4a) and b. 4CR-2CP pathway (li, 2c, 3o, and 4a). c. E21 -labeled insulin for bioactivity assay: Western blot analysis of pAkt and P-actin in HEK293T cell lysates, d. Quantification of pAkt signal relative to P-actin. e. Detection of insulin signaling pathway activation (pAkt-S473 level, red) upon treatment with native (li) or E21- labeled insulin (69) in HeLa cells. Internal control P-actin (green) and chromatin (blue) (scale bar: 10 pm) are also stained, f. 4CR-2CP pathway (Ij, 2f, 3g, and 4a) enabled synthesis of homogeneous trastuzumab conjugate (71, LC: D28; HC: D62, E336, E321). The late-stage installation of fluorophore (FITC, 72) or drug (emtansine, DBC0-DM1, 73) renders 4CR-2CP-AFC (74) and 4CR-2CP-ADC (75). g. Antigen-specific cell surface binding and uptake of AFC 74 (green) in SKBR-3 cells, h. Inhibition of cell proliferation by DM1 (76), 4CR-2CP-ADC (75), T-DM1 or Kadcyla (77), and trastuzumab (Ij) in SKBR-3 (HER-2 positive) cancer cell line. The %inhibition is calculated by using untreated cells as control, i. Inhibition of cell proliferation by DM1 (76), 4CR-2CP-ADC (75), Kadcyla (77), and trastuzumab (Ij) at 1 nM in HER-2 positive SKBR-3 as compared to HER-2 negative MDA-MB-231 cells (scale bar: 10 pm). Data are presented as mean values (±SD), n = 3 biologically independent experiments, in accordance with the present invention;

[0046] Figure 8: illustrates a) synthesis of compound 79 via multicomponent reaction of (S)-4-((tert-butoxycarbonyl)amino)-5-methoxy-5-oxopentanoic acid (78), phenylhydrazine hydrochloride (2k), formaldehyde (3a), and tert-butyl isonitrile (4a) in 40% aqueous acetonitrile at ambient temperature (25 °C) for 24 h. The product is purified by column chromatography and isolated in 34% yield, b) Selective modification of Glu in model peptides by using the reagents 2k, 3a, and 4a. Peptides containing Glu and a variable amino acid at the i and i + 4 positions showed site- selective modification at Glu (85-89). In peptides with Asp at i and Glu at i + 4, modification occurred preferentially at the Glu residue (90), in accordance with the present invention;

[0047] Figure 9: illustrates a) pathway-selective, chemoselective, and site-selective 4CR- 4CP with diverse proteins, b) Heterogeneous labelling of a diverse set of proteins via the 4CR-4CP pathway. Despite structural variability among proteins, the labelling proceeds through a consistent reaction mechanism across all cases, c) Functional group diversification - installation of chemically orthogonal handles (102, 103), probes (104, 105), in accordance with the present invention; and

[0048] Figure 10: illustrates the a) Evaluation of pathway selectivity of the 4CR- 4CP reaction in a complex biological environment using a mixture of four distinct proteins, b) Total ion chromatogram (TIC) of the protein mixture. Mass spectrometric analysis confirmed that the 4CR-4CP pathway maintained its selectivity within the heterogeneous protein mixture, in accordance with the present invention.

[0049] DETAILED DESCRIPTION OF THE INVENTION The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the present disclosure as defined by the appended claims.

[0050] In an embodiment, the present invention discloses a complex multi-equilibria system with multiple reaction pathways, achieving highly selective outcomes even with complex substrates like proteins. The present invention offers a comprehensive method for achieving highly selective outcomes, including exclusive control over specific pathways like the four-component reaction yielding a four-component product (4CR-4CP), along with the ability to switch pathways entirely while maintaining selectivity even at high conversions (>99%).

[0051] Further, the present invention allows exclusive control over chemoselectivity, siteselectivity, and modularity, enabling single-site modification in over thirty-six examples. The present invention not only introduces a new dimension to chemical engineering but also challenges established principles of reactivity and selectivity. It offers a comprehensive framework with three methods for single-site modification of carboxylates in proteins. Furthermore, the present invention demonstrates the translation of these methods for dual-probe engineering, including FRET pairs, enabling precise carboxylate engineering of insulin and access to homogeneous antib ody-fluorophore and drug conjugates (AFC and ADC). The present invention leverages multi-equilibria systems to harness biomolecular complexity for precision engineering.

[0052] In an embodiment, the method of the present invention is as follows:

[0053] Multi-equilibria systems

[0054] In this embodiment, the present invention explores the potential of multi-equilibria systems by utilizing proteinogenic functionalities to generate a range of intermediates, as depicted in Figure 1. Lysozyme C (la), primary amine (2a), aliphatic aldehyde (3), and isonitrile (4) (as shown in Fig. 2a) are vortexed together. The resulting N-imine formed reacts with an adjacent amide, yielding Lys-imines (5a) that further react with proximal nucleophilic residues. Additionally, these imines can react with isonitrile, forming an electrophilic intermediate (path I, 5b) that hydrolyzes easily to form a 3 CP (5d, path I, Fig. 1).

[0055] The aldehyde functional group may form an iminium species either with a tryptophan (Trp) residue via an intramolecular pathway (Path II, intermediate 6a), or with an external imine species (intermediate 7a). These intermediates are capable of undergoing further reaction with a tyrosine (Tyr) residue (Path III), resulting in irreversible transformation to product (7c). Furthermore, the carboxylate gives rise to an O-acyl imidate intermediate (intermediate 8c), which facilitates an irreversible [l,3]-O,N-acyl transfer, yielding a four-component reaction-four-center product (4CR-4CP, intermediate 8d, Path IV). In an alternative pathway (Path V), the carboxylate group may displace the amine, leading to the formation of an imidate intermediate (intermediate 9b), which subsequently rearranges to generate the three- center product (3CP, intermediate 9c).

[0056] Furthermore, an additional reaction pathway may result from the electrophilic formate species formed via oxidation of an aldehyde, which can subsequently lead to the formulation of lysine (Lys) residues. These representative pathways illustrate the intricate system of multiple equilibria that exists within protein structures, wherein the selection of specific reaction pathways is influenced by the particular combination of reagents employed.

[0057] Pathway selection by proteins

[0058] Furthermore, lysozyme C (la) vortexes with benzyl amine hydrochloride (2a), formaldehyde (3a), and t-butyl isonitrile (4a) for a control experiment (Fig. 2a). The reaction yielded a diverse array of products, including 4CR-3CP (10a, 4% yield), 4CR-4CP (10b: 26% mono-, 19% bis-, and 3% tris-labelled), and combinations of both within the same protein bioconjugate (10c: 8% mono- and 5% bis-labelled, each). An unstable la-3a-4a adduct (lOd: 25% mono- and 9% bis-labelled) is observed. The protein sequencing confirmed modification of Lys, Glu, and Asp in 4CR-3CP via distinct pathways (KI, E7, E35, and DI 19), while 4CR-4CP exhibited chemoselective modification of Asp (D101, DI 19, D52, D18, D48). While the selectivity was suboptimal, the results substantiate the feasibility of pathway discrimination and targeted bioconjugation.

[0059] To explore further, the present invention utilized a sterically more demanding aldehyde (n-butyraldehyde, 3b) while maintaining the other components constant (la, 2a, and 4a, Fig. 2b). This resulted in the formation of a 4CR-3CP [I la, 10% mono-labelled, path I], 4CR-4CP [11b, 4% mono-labelled, path IV], and an unstable la-3b-4a adduct [11c, 26% mono-labelled]. The sequencing confirmed the selective modification of KI (4CR-3CP) and D66 (4CR-4CP) products.

[0060] 4CR-4CP pathway selectivity, chemoselectivity, and site-selectivity

[0061] The above findings validate the regulation of pathway-selectivity in conjunction with chemoselectivity and site-selectivity. The present invention discloses that rapid aldehyde consumption restricts the participation of proteinogenic amines (5a, path I, Fig. 1). Unfortunately, excess amine in the Ugi reaction failed to address this issue. To overcome this limitation, the present invention replaces the amine with hydroxylamine hydrochloride (2b) for in-situ oxime formation (8a, Fig. 1). Remarkably, combining la with 2b, 3a, and 4a completely suppresses the 4CR-3CP pathway, leading exclusively to the 4CR-4CP pathway (12, 47% mono-labelled). Sequencing confirmed homogeneous D52 modification. This confirms the feasibility of achieving exclusive pathway selection alongside chemoselectivity and siteselectivity, even resulting in single-site carboxylate modification, a highly challenging residue for protein bioconjugation.

[0062] The impact of diverse aldehydes on selectivity is demonstrated (Fig. 2c). The 4CR- 4CP pathway remained unchanged in the presence of n-butyraldehyde (3b) and isobutyraldehyde (3c). However, aromatic aldehydes (3d, 3e, and 3f) did not lead to any irreversible bioconjugation. Subsequently, the present invention investigated the reactivity of a range of isonitriles 4b-d with la, 2b, and 3a (Fig. 2d). Furthermore, the optimal 4CR-4CP protocol (2b, 3a, and 4a) is applied to a structurally diverse set of proteins. In one embodiment, RNase A (lb) exhibited exclusive 4CR-4CP pathway selectivity and chemoselectivity, labeling three carboxylates (17: 17% mono- and 13% bis-labeled). Ubiquitin (1c) similarly displayed selectivity, resulting in 4CR-4CP (18: 21% mono-, 20% bis-labeled). Finally, the protocol applied to cytochrome C (Id) resulted in single-site E69 modification (19, 4CR-4CP, 18% mono-labelled).

[0063] Re-routing 4CR-4CP to 4CR-2CP

[0064] In the preceding step, the steric considerations of aliphatic aldehydes maintained the selection of the 4CR-4CP pathway (Fig. 2c). To explore the steric influences of hydroxylamine in conjunction with aldehyde regulation on pathway preference, O- benzyl hydroxylamine hydrochloride (2c) is utilized alongside la, 3a, and 4a. Interestingly, it is observed that alongside the predominant 4CR-4CP pathway (20a: 38% mono-, 17% bis-, and 3% tris-labelled), the 4CR-2CP pathway emerged (20b, 8% mono-labelled), along with a combination of 4CR-4CP / 4CR-2CP (20c, 7% mono-labelled) (Fig. 3a). The 4CR-4CP pathway involved four Asp residues (D48, D52, D87, and DI 19), whereas the 4CR-2CP pathway engaged two Asp residues (D52 and DI 19). The unequivocal identification of the 4CR-2CP pathway confirms the possibility of switching pathway selectivity. This represents the first proof of principle for interm olecular reaction (21e) surpassing an intramolecular reaction (21d, Fig. 3b).

[0065] Multi-equilibria disruption for 4CR-2CP

[0066] In another embodiment of the present invention, a screening of diverse aldehydes (3) is conducted in conjunction with t-butyl isonitrile (4a), (9-benzylhydroxylamine hydrochloride (2c), and lysozyme C (la). Gratifyingly, n-butyraldehyde (3b) yielded exclusive 4CR-2CP pathway selectivity alongside chemo- and site-selectivity (22, 38% mono-labeled, Fig. 4a). Sequencing confirmed single-site DI 19 modification. As anticipated, a few other aldehydes (3c, 3d) exhibited similar pathway selectivity (22, Fig. 3c). Furthermore, to explore the translation to aromatic aldehydes, it is found that 2-hydroxybenzaldehyde (3e), benzaldehyde (3f), and 3,4- dihydroxybenzaldehyde (3g) resulted in single-site labeling through the 4CR-2CP pathway.

[0067] Subsequently, the present invention established that selectivity attributes remained consistent with diverse isonitriles (4b-4d) in the presence of la, 2c, and 3b (Fig. 3d).

[0068] 4CR-2CP with diverse proteins

[0069] Further optimization of the protocol is conducted with various proteins: RNase A (lb, 5 Asp, 5 Glu, and IC-terminal) using 2c, 3b, and 4a (Fig. 4a) resulted in a homogeneous single-site labelled bioconjugate (26, D121, 30%). Ubiquitin (1c, 5 Asp, 6 Glu, and 1 C-terminal) exhibited exclusive 4CR-2CP selection, yielding a D52-labeled bioconjugate (27, 38%). Cytochrome C (Id, 3 Asp, 9 Glu, and 1 C- terminal) also produced a homogeneous D2-labeled bioconjugate (28, 24%). Myoglobin (le, 8 Asp, 13 Glu, and 1 C-terminal) exhibited exclusive DI 09-labelling (29, 26%). Conversely, a-lactalbumin (If, 13 Asp, 7 Glu, and 1 C-terminal) resulted in El l-labelled 4CR-2CP (30a, 30%), partially accompanied by the 4CR-4CP pathway (30b, 4% mono-labelled).

[0070] To enhance site-selectivity, serum albumins are selected. Bovine serum albumin (BSA, 1g), comprising 39 Asp, 58 Glu, and 1 C-terminal carboxylic acid, yielded a 4CR-2CP pathway-selective, mono-labeled, yet site-heterogeneous bioconjugate (31, 41%, D258 and D268). Conversely, Human Serum Albumin (HSA, Ih, 34 Asp, 64 Glu, and 1 C-terminal) reaction with 2c, 3b, and 4a resulted in exclusive 4CR-2CP pathway selectivity alongside chemoselectivity, site- selectivity, and sitehomogeneity (E252, 32, 42%). These results validate the excellent control over selectivity attributes with the combination of 2c, 3b, and 4a. However, minor compromises in pathway selectivity or site-homogeneity may occur in some cases.

[0071] Examining the capabilities of 3,4-dihydroxybenzaldehyde (3g) in the multi-equilibria system is also explored.

[0072] Aldehyde enabled modularity in 4CR-2CP Utilizing 3,4-dihydroxybenzaldehyde (3g), an initial screening is conducted with la, 2c, and 4a (Fig. 4b). This yielded single-site D18-labeling with exclusive 4CR-2CP pathway (23, 40%). Subsequently, diverse isonitriles (4b-4d) are screened. While the pH range of 6.0-7.0 proved ideal for single-site bioconjugation, it is observed to cease at pH 7.5 or above, indicating dependence on buffer composition and concentration.

[0073] Finally, it is established that the 4CR-2CP pathway selectivity remains consistent across a broad spectrum of aromatic and heteroaromatic aldehydes.

[0074] 4CR-2CP with diverse proteins

[0075] In one embodiment of the present invention, optimized conditions are established for structurally diverse proteins. Altering the aldehyde (3b versus 3g, Fig. 4a versus 4b) preserved the 4CR-2CP pathway and chemoselectivity while introducing modularity. Initially, changing the conjugation site on lysozyme C (la) from D52 to D18 illustrated this modularity (Figs. 4a and 4b). Vortexing RNase A (lb) with 2c, 3g, and 4a (Fig. 4b) resulted in exclusive 4CR-2CP pathway selectivity and single-site modification at D121 (33, 35%).

[0076] For ubiquitin (1c), the 4CR-2CP pathway selectivity (34, 45%) accompanied a site change to D58. Similar modularity was observed with cytochrome C (Id), resulting in D50 labeling (35, 22%). Myoglobin (le) showed labeling at D141 (36, 24%) instead of D109. In a-lactalbumin (If), the labeling site changed from El l to D64 (37, 30%). Extending the methods to serum albumins, BSA (1g) demonstrated exclusive labeling of E229 instead of D258 / D268 (38, 41%) with notable site homogeneity. Applying the method to HSA (Ih) resulted in E230 modification (39, 43%) instead of E252.

[0077] These results confirm that multi-equilibria systems offer excellent control over chemoselectivity, site-selectivity, and modularity, with exclusive 4CR-2CP pathway selectivity.

[0078] To evaluate the impact of 4CR-2CP on the structure and activity of the proteins, the structure of 4CR-2CP bioconjugates remained unchanged (23 and 31-37). Specifically, D18-labeled lysozyme C (23) retained its activity towards Micrococcus lysodeikticus, confirming that its activity remained unaltered post-bioconjugation.

[0079] Re-routing 4CR-2CP to 3CR-2CP

[0080] Continuing the examinations, it is hypothesized that a proteinogenic carboxylate reacts with an isonitrile to generate an electrophilic intermediate (40, Fig. 5a). The presence of this intermediate is confirmable by trapping it with an external nucleophile, potentially resulting in a 3CR-2CP pathway (42). Although additional pathways (41 or 43) may introduce complexity, the potential to expand the targetome by labeling sites distinct from the 4CR-2CP pathway remains of significant interest. To investigate this, lysozyme C (la) is vortexed with 4a and 2c (Fig. 5b). Complete control over chemoselectivity, site-selectivity, and 3CR-2CP pathway selectivity is observed (44, 35%). The modification site is identified as D48, which differs from those observed in the 4CR-2CP pathway (DI 19 with 3b and D18 with 3g), thereby demonstrating the utility of the multi-equilibria system as a platform for modular and precise protein engineering methods.

[0081] Subsequently, various attributes are screened, including the effect of isonitriles (4), pH, buffer composition, and protein concentration (44). To evaluate the tolerance of pathway-selectivity at higher conversions, lysozyme C (73 pM), 2c (73 mM), and 4a (73 mM) are incubated for 8 hours, resulting in >96% conversion to the bioconjugate (44). Despite labeling up to six sites, the 3CR-2CP pathway and chemoselectivity remained uncompromised. Following parameter tuning, the optimal protocol is selected to examine the conservation of pathway selectivity in structurally diverse proteins.

[0082] Initially, RNase A (lb) results in single-site D121 modification (45, 25%, Fig. 5b). Similarly, ubiquitin (1c) vortexed with 2c and 4a demonstrated exclusive 3CR-2CP pathway selectivity (46, 33%), providing access to a novel targetome. This trend extended to cytochrome C (Id), yielding a mono-labeled bioconjugate (D50 / E103, 47, 22% conversion, Fig. 5b). Myoglobin (le) offered exclusive 3CR-2CP and access to a new domain with D20-labeling (48, 18% conversion). Likewise, a-lactalbumin (If) rendered E49-labeled 3CR-2CP (49, Fig. 5b), distinct from El l or D64 in 4CR- 2CP. This modularity further extends to bovine serum albumin (BSA) (1g) with D493 modification (50, 39% conversion). Finally, HSA (Ih) resulted in 3CR-2CP (E16 / D269, 36%, 51) with sites distinct from 4CR-2CP. Consistent with earlier findings; 3CR-2CP modifications did not perturb the protein structure (44-51). Additionally, the established D48-labeling didn’t affect lysozyme C activity (44). Overall, this validates that 3CR-2CP offers a new carboxylate targetome with conserved bioconjugate structure and function.

[0083] Precision engineering of chemically orthogonal handles, probes, and bond architectures

[0084] To demonstrate the synthetic flexibility of the present method, diverse hydroxylamine derivatives (2d-2j, Fig. 5c) are employed in conjunction with 3CR- 2CP. Combining lysozyme C (la) with 4a and an azide-equipped hydroxylamine (2d) resulted in the exclusive formation of a homogeneous bioconjugate labeled at D48 (52, 35%, Fig. 5c). Similarly, an alkyne handle (2e) is selectively incorporated through the 3CR-2CP pathway (53, D52, 36%). The method seamlessly extended to equip the bioconjugate with both alkyne and azide handles (54, D48, 36% monolabeled). Likewise, the 3CR-2CP pathway selectivity remained consistent during the direct installation of a fluorophore (55, DI 19, 30%) or a 19F-NMR probe (56, D48 / D52, 34%).

[0085] In exploring whether altering the external nucleophile provides access to diverse bond architectures, it is found that the 3CR-2CP pathway selectivity remains unchanged upon switching to N-benzylhydroxylamine hydrochloride (2i), resulting in a single-site labeled bioconjugate (57). Similarly, the use of hydrazine hydrochloride (2j) yields unique bond architecture (58).

[0086] Dual probe protein engineering and biophysical investigations

[0087] Precisely engineering of dual-probe protein bioconjugates are crucial for Forster Resonance Energy Transfer (FRET)-based assays. In another embodiment, the 3CR- 2CP pathway enabled the synthesis of E45-labeled BSA (59, 41%). Subsequently, 59 are vortexed with DBCO-Cy5 (60) for the C34-thiol-yne reaction (61). The absorption and emission spectra of the protein, individually labeled by Cy3 (63) and Cy5 (61), are depicted in Fig. 6b. A significant spectral overlap for efficient FRET is established from the normalized plot of the donor (63) emission and the acceptor (61) absorption. The fluorescence spectra of 63 are recorded by exciting the sample at the Cy3 excitation wavelength (550 nm), revealing a peak centered at -670 nm as a FRET signature (Fig. 6c).

[0088] Yet in another embodiment, time-resolved fluorescence experiments are conducted to determine the lifetime before and after dual labeling of the protein (1g and 64). The decrease in lifetime confirmed efficient FRET (EFRET, 54.2%) between Cy3 and Cy5 installed at two sites of the protein (64, Fig. 6d). Specifically, the average excited-state lifetime of the donor (E45-Cy3, 63) decreased from 1.27 ns to 0.58 ns upon labeling with the acceptor (C34-Cy5, 61).

[0089] Yet in another embodiment, Bovine Serum Albumin (BSA) is C-terminally labeled in the presence of compounds 2f and 4a, affording the corresponding bioconjugates in 65% and 38% yields, respectively. Subsequently, DBCO-Cy5 (compound 60) and azide-functionalized Cy3 (compound 62) are installed, yielding bioconjugates 66 and 67. These conjugates are further subjected to sequential installation of DBCO-Cy5 and azide-equipped Cy3 to afford the dual -labeled construct C-ter-Cy5 / C-ter-Cy3- BSA (compound 68, as depicted in Fig. 6e). The structural integrity of compound 68 is confirmed via spectroscopic analysis. The steady-state spectroscopy of Cy3 (67) and Cy5 (66) labeled protein is performed. Subsequently, the recorded fluorescence spectra of the single-site dual-labeled BSA (68, >EX = 550 nm), exhibiting an emission maximum at -670 nm (Fig. 6f), corresponding to Cy5 (acceptor) emission, inferring FRET between the two fhiorophores installed at a single site. Further, a significant reduction in the excited state lifetime of the donor fluorophore substantiates the conclusion drawn from steady-state emission that the FRET process culminates from the dually labeled C-terminal (Fig. 6g). The FRET efficiency estimated from the excited state lifetime of the donor (54.3%) confirms significant energy transfer and proximity between the two fluorophores.

[0090] Precise and modular engineering of insulin The advancement of chemical technology for carboxylate modification in Insulin is highly anticipated. However, the present method for precision engineering of carboxylates remains limited. Therefore, the present method extends the 3CR-2CP pathway to insulin, wherein reaction with components 2c and 4a yields E21 -labeled insulin (compound 69) with 36% mono-labeled product, as shown in Figure 7a. Further application of the 4CR-2CP pathway using insulin (li) in combination with 2c, 3g, and 4a achieves high site-selectivity, albeit at the same E21 residue (Figure 145). Notably, implementation of the 4CR-2CP pathway with component 3o induces a site-selectivity switch from the E21 residue to the C-terminal, affording compound 70 in 36% yield (Figure 7b).

[0091] To evaluate the effect of the modification on structural integrity, receptor binding, and downstream signalling activity, compound 69 is selected (Fig. 7c). The treatment of the cells with compound 69 and its unmodified counterpart (compound li) results in an increase in pAkt levels (Fig. 7d). The magnitude of pAkt-S473 signals observed in HEK293T cell lysates is comparable for both compounds, as determined by western blot analysis (Fig. 6c). Additionally, E21 -labeled insulin (compound 69) induces intracellular pAkt levels in HeLa cells that are comparable to those induced by the unmodified insulin (visualized as red signals in the first vertical panels, Fig. 7e). These results collectively indicate that the modification does not impair receptor binding or downstream signaling activation of the insulin molecule.

[0092] Antibody Conjugates with Fluorophore and Drug (AFC and ADC)

[0093] Yet in another embodiment of the present invention, a method for synthesizing antibody conjugates is described. The 4CR-2CP reaction is extended to prepare the antib ody-fluorophore conjugates (AFCs) and antibody-drug conjugates (ADCs) (Fig. 7f). Trastuzumab (Ij) is treated with 2f and 4a compounds to install chemically orthogonal functional handles (71, azide and alkyne) achieving a probe-to-antibody ratio of ~1.5. Site-specific labeling is observed, wherein the light chain is modified at residue D28, and the heavy chain is modified at residues D62, E336, and E321. These modifications facilitate the subsequent attachment of a fluorophore (FITC- azide, 72) or a cytotoxic drug (DBC0-DM1, 73), yielding the corresponding AFC (74) and ADC (75), respectively. The stability of AFC (74) is evaluated in 10% human serum over a period of seven days, demonstrating 95% retention of the conjugate. The antigen targeting capability of AFC (74) is confirmed via immunofluorescence analysis on HER-2-positive SKBR-3 cells and HER-2-negative MDA-MB231 cells. The fluorescence is detected exclusively in SKBR-3 cells, indicating antigen-specific targeting (see Fig. 7g).

[0094] Furthermore, ADC (75) is evaluated in a cytotoxicity assay using HER-2- overexpressing SKBR-3 cells. A dose-dependent inhibition of cell proliferation is observed, with 47% inhibition at a 1 nM concentration of ADC (75), a result comparable to that obtained with DM1 (76, emtansine). Additionally, ADC (75) demonstrates significantly greater efficacy compared to Kadcyla (77), which exhibits only 26% inhibition at the same concentration. Notably, ADC (75) exhibits no measurable cytotoxic effect on HER-2-negative MDA-MB231 cells under identical conditions, whereas treatment with 1 nM DM1 alone results in 18% inhibition (see Fig. 7j). These findings establish that a homogeneous carboxylate-engineered ADC provides enhanced therapeutic efficacy at reduced drug loading, specifically toward antigen-expressing target cells.

[0095] Hydrazine based pathway

[0096] In embodiment of the present invention, the hydrazine-based bioconjugation pathway employs a strategic selection of reagents to enable the incorporation of bio- orthogonal handles, purification tags, and imaging tags within a single bioconjugation step, specifically targeting a predetermined residue on the biomolecule. In this embodiment, hydrazine hydrochloride serves as a core reagent due to its high nucleophilicity and capacity to form stable intermediates, thereby enhancing the overall efficiency of the transformation. The method commenced with the reaction of (S)-4-((tert-butoxycarbonyl)amino)-5-methoxy-5-oxopentanoic acid (78), phenyl hydrazine hydrochloride (2k), formaldehyde (3a), and tert-butyl isonitrile (4a) in 40% aqueous acetonitrile at ambient temperature (25°C) for 24 h. Following completion of the reaction, the product is subjected to purification via column chromatography and isolated in 34% yield (Fig. 8a). the High-resolution mass spectrometry (HRMS) analysis confirms the formation of the product. To further validate the chemoselectivity of the process, the method is subsequently applied to model peptides, as shown in Figure 8b.

[0097] Yet another embodiment of the present invention, a panel of model peptides comprising glutamic acid (Glu, E) as the target residue is designed (Fig. 8b). To ensure steric accessibility of the reactive center, alanine (A) and glycine (G) residues are positioned adjacent to the Glu residue. Furthermore, two arginine residues are incorporated to enhance peptide solubility. A series of peptides is synthesized to evaluate the efficiency of solid-phase peptide synthesis across different sequence contexts. The peptide H-AEGRRK-NFE is obtained in a moderate yield of 43%, while H-AEGRRH-NEE is obtained in a yield of 39%, indicating comparable synthetic efficiency. The sequence H-AEGRRS-NH2 exhibits a slightly higher yield of 54%, and H-AEGRRM-NH2 demonstrates a significantly enhanced synthetic efficiency with a yield of 99%.

[0098] In all tested sequences, selective modification at the Glu residue is observed, with no detectable modification at other nucleophilic residues, thereby demonstrating the chemoselectivity of the reagent (Fig. 8b). Additionally, in a model peptide comprising both Glu and aspartic acid (Asp, D), the reagent exhibits preferential reactivity toward Glu over Asp, indicating a high degree of selectivity even among structurally related carboxylate residues. These observations collectively establish the reagent’s robust chemoselectivity under the tested reaction conditions.

[0099] A mixture comprising a protein (1), a hydrazine compound (2), an aldehyde (3), and an isonitrile (4) affords multiple reactive combinations, resulting in the formation of a structurally diverse pool of intermediates (Fig. 1). The optimization of reaction parameters is essential to maximize the efficiency and selectivity of the desired bioconjugate. The control experiments confirm the formation of the 4CP product via the proposed reaction pathway.

[0100] Among various buffer systems evaluated, HEPES buffer facilitates kinetically controlled access to the 4CP product, affording a mono-labeled conjugate in 24% yield. In contrast, alternative buffer systems shift the reaction toward faster, less selective pathways. When the reaction is conducted at pH 7.0, formation of the 4CP product occurs with a conversion of 24%. A systematic evaluation of pH conditions reveals that the 4CP pathway remains exclusive across a broad pH range, although the efficiency of conversion varies. However, at pH values exceeding 7.5, a competing 4CR-2CP pathway emerges.

[0101] Protein concentration is subsequently optimized in the range of 2 to 10 nmol, with 7.3 nmol providing the highest conversion efficiency of 24% to the mono-labeled 4CP product. The variation of the hydrazine hydrochloride equivalents identifies 200 equivalents as optimal for efficient 4CP formation. Temperature is also found to be a critical factor; selective formation of the 4CP product is achieved at 25°C, whereas elevated temperatures promote the formation of a competing 4CR-3CP product.

[0102] The solvent composition significantly influences the outcome of the bioconjugation. To preserve protein structural integrity and maintain reaction selectivity, 20% acetonitrile (ACN) is determined to be optimal for mono-labeled 4CP bioconjugate formation. The lower percentages of ACN result in increased formation of byproducts via 4CR-3CP and 4CR-4CP pathways.

[0103] The potential influence of hydrazine hydrochloride on the reaction pH is assessed using control experiments employing hydrazine, hydrazine hydrochloride, and hydrazine in combination with hydrochloric acid. These experiments reveal minimal deviation from the designated buffer pH of 7.0.

[0104] To further refine the scope of the reaction, a series of aldehydes is evaluated. Among these, the aliphatic aldehyde formaldehyde (3a) consistently promotes the formation of the 4-component product (4CP) under optimized conditions, resulting in irreversible bioconjugate formation (Figure 9a). Other aldehydes do not yield the 4CP within 15 minutes under comparable conditions. Additionally, a library of isocyanide derivatives is screened for their efficiency in facilitating 4-component reaction (4CR)-4CP bioconjugation of insulin. All reactive isocyanides direct conjugation selectively to residue El 7. Among them, tert-butyl isocyanide provides the highest conversion (24%), followed by cyclohexyl (22%) and n-butyl (10%) isocyanides, all targeting El 7. Morpholine-based isocyanide fails to produce a stable conjugate. These results indicate that while the structure of the isocyanide influences the conversion efficiency, the site of labeling remains consistent across all active derivatives. The optimal reagent combination for exclusive 4CP formation comprises tert-butyl isocyanide (4a), phenylhydrazine hydrochloride (2k), and formaldehyde (3 a).

[0105] To monitor the progression of insulin labeling over time via the hydrazine-mediated multicomponent reaction, time-course studies are conducted. The percentage conversion of insulin into mono-, bis-, tris-, tetra-, and penta-labeled forms is determined at defined time intervals. Mono-labeled insulin forms rapidly and increases progressively, exceeding 40% conversion at 120 minutes. As the reaction proceeds, higher-order labeled species emerge, particularly beyond 180 minutes, indicating that the labeling occurs in a stepwise manner. Residue-level reactivity mapping reveals that residue El 7 of chain A undergoes initial modification, followed by subsequent labeling at the C-terminal of chain A and additional residues at later time points. These temporal studies establish the order of site reactivity and demonstrate that the 4CR-4CP labeling strategy affords both site selectivity and temporal control over conjugation.

[0106] The robustness of the optimized protocol is validated across a panel of proteins exhibiting diverse structural and functional characteristics, targeting accessible carboxylate side chains. The proteins include lysozyme C (la), RNase A (lb), human serum albumin (HSA, Ih), myoglobin (le), and insulin (li). Each protein affords selectively mono-labeled conjugates, with the respective labeling sites confirmed by peptide mapping. Insulin and myoglobin are modified at residues E17 and E18 with conversion efficiencies of 24% and 27%, respectively. HSA provides the highest conversion efficiency of 42% at residue E321, which is attributed to increased conformational flexibility and solvent exposure of the reactive site. Lysozyme C and RNase A are labeled at residues D52 and E2, respectively, with conversion efficiencies of 26% and 19%, demonstrating the method's compatibility with both aspartate and glutamate residues. The 4CR-4CP strategy exhibits high chemoselectivity and broad protein compatibility, thereby establishing its utility for site- selective labeling of carboxylate-containing side chains. Even under conditions yielding greater than 99% labeling efficiency, pathway selectivity remains exclusive to the 4CP route. Structural analyses confirm that the conformations of the proteins remain unaltered following conjugation, thereby supporting the non-disruptive nature of the 4CR-4CP modification. Furthermore, the biological activities of lysozyme C, HSA, and myoglobin remain comparable to their native forms post-labeling. Specifically, despite successful modification of residue D52 in lysozyme C, its catalytic activity remains substantially unaltered.

[0107] To evaluate whether pathway selectivity is retained in complex biological environments, a protein mixture comprising four distinct proteins-Lysozyme C (la), RNase A (lb), myoglobin (le), and insulin (li) is assembled. The mass spectrometric analysis confirms that the selectivity of the 4CR-4CP pathway remains intact within this heterogeneous protein mixture (see Fig. 10). These findings indicate that increased protein complexity does not compromise pathway selectivity. On the contrary, such complexity facilitates a more refined and controllable bioconjugation process, consistent with the preservation of selectivity observed in crowded cellular environments.

[0108] Following validation of the multi-equilibria disruption strategy, insulin is engineered to incorporate bio-orthogonal functional groups. Hydrazine hydrochloride salts are initially synthesized to evaluate their reactivity within the multicomponent reaction framework. Compounds 21 and 2m are designed to demonstrate the chemical versatility of the hydrazine moiety. However, these substrates do not yield detectable levels of conjugation under the tested conditions. To address this limitation, alkyne- and azide-functionalized probes lacking heteroaromatic rings are synthesized. These probes enable site-selective modification of insulin under multi-equilibria reaction conditions. Probe 2o achieves a 22% conversion at residue E21 (105, Fig. 9c), and probe 2n achieves a 19% conversion at the C-terminus (104, Fig. 9c). Additionally, probes 21 and 2m, containing alkyne and azide functionalities respectively, afford 13% and 12% conversion, both targeting residue E21 (102-103, Fig. 9c). Chemical diversification is further demonstrated by incorporating a19F-NMR probe (2n) and a Dansyl fluorophore (2o) at residue E21 and the C-terminus, respectively (Fig. 9c). Incorporation of the19F tag is confirmed via19F NMR spectroscopy. All modifications are accomplished in a single-step reaction, yielding structurally defined and multifunctional insulin bioconjugates. The disclosed hydrazine-based 4CR-4CP conjugation strategy demonstrates significant potential for broad biopharmaceutical applications, including but not limited to the rapid generation of modified therapeutic proteins, such as fast-acting insulin analogs. As a proof-of- concept, the methodology was applied to a clinically relevant monoclonal antibody, trastuzumab, to assess the chemo-selectivity and compatibility of the conjugation platform with complex protein architectures. Site-specific modifications were evaluated through light and heavy chain analyses, as well as tandem mass spectrometry (MS / MS), which collectively confirmed selective modification at carboxylic acid residues. These findings not only validate the applicability of the platform to therapeutically relevant antibodies but also provide a foundation for future developments where selective carboxylic acid engineering could be leveraged for conjugation strategies, such as in the construction of antibody conjugates with enhanced functional outcomes.

[0109] ADVANTAGES OF THE PRESENT INVENTION

[0110] An advantage of the present invention is to provide unprecedented precision in the engineering of biomolecules.

[0111] An advantage of the present invention is to enable selective modification of specific sites on biomolecules.

[0112] An advantage of the present invention is to provide flexibility for the synthesis of diverse bioconjugates.

[0113] An advantage of the present invention is to facilitate the development of novel therapeutics and diagnostics.

[0114] An advantage of the present invention is compatible with a wide range of biomolecules and reaction conditions. Although a preferred embodiment of the invention has been illustrated and described, it will at once be apparent to those skilled in the art that the invention includes advantages and features over and beyond the specific illustrated construction. Accordingly, it is indented that the scope of the invention be limited solely by the scope of the hereinafter appended claims, and not by the forgoing specification, when interpreted in light of the relevant prior art.

Claims

AMENDED CLAIMS received by the International Bureau on 17 November 2026 (17.11.2025)We Claim:

1. A method for site -selective bioconjugation of proteins via a multi -equilibria disruption mechanism utilizing one or more pathways, comprising of:(1) a four-component reaction leading to a four-component product (4CR-4CP),(2) a four-component reaction leading to a two-component product (4CR-2CP), or(3) a three-component reaction leading to a two-component product (3CR-2CP); wherein the method comprises:(a) providing a protein comprising at least one proteinogenic carboxylate side chain;(b) reacting said protein with:(i) an aldehyde compound selected from aliphatic, aromatic, or heteroaromatic aldehyde of Formula (3);(ii) an isonitrile compound of Formula (4), selected from tert-butyl isonitrile, cyclohexyl isonitrile, and n-butyl isonitrile; and(iii) a nucleophile selected from hydroxylamines, hydrazines and primary amines of Formula (2);(c) conducting the reaction under controlled conditions of pH 6.0-7.0, temperature 4-37 °C, and solvent composition containing 0-20 % organic solvent in aqueous buffer selected from Tris or HEPES or PBS, thereby forming an equilibrium system;(d) isolating a site-selectively labeled protein conjugate having modification at one or more carboxylate residues, wherein the pathway (a-c) is determined by the aldehyde, isonitrile, and nucleophile combination.

2. The method as claimed in claim 1, wherein the aldehyde is selected from aliphatic aldehydes selected from n-butyraldehyde (3b), aromatic aldehydes selected from 2-hydroxybenzaldehyde (3e), benzaldehyde (3f), 3,4-dihydroxybenzaldehyde (3g), and heteroaromatic aldehydes selected from N, O, or S within an aromatic ring.

3. The method as claimed in claim 1, wherein the protein is selected from the group consisting of RNase A, ubiquitin, cytochrome C, myoglobin, a-lactalbumin, bovine serum albumin (BSA), human serum albumin (HSA), and monoclonal antibodies including trastuzumab, exhibits site-specific labelling of D28 (light chain) and D62, E336, E321 (heavy chain).

4. The method as claimed in claim 1, wherein site -selective carboxylate modification occurs at a residue selected from DI 19 or D18 of lysozyme C, D121 of RNase A, D52 of ubiquitin, D2 of cytochrome C, DI 09 of myoglobin, El l of a-lactalbumin, D258 or D268 of BSA, or E252 of HSA.

5. The method as claimed in claim 1, wherein the protein is a model peptide containing a single carboxylate residue, and only the carboxylate residue is modified.

6. The method as claimed in claim 1, wherein the protein is insulin, and the modification occurs selectively at residue El 7 or E21, yielding a mono-labeled insulin bioconjugate retaining receptor-binding and signalling activity.

7. The method as claimed in claim 1, wherein the protein is bovine serum albumin (BSA) and the first fluorophore is Cy3 while the second is Cy5, generating a C34- Cy5 / E45-Cy3 dual-labelled conjugate.

8. The method as claimed in claim 1, wherein the reaction is conducted at a protein concentration of 70 to 80 pM, with aldehyde and isonitrile at 70 to 80 mM, for a duration of about 6-10 hours, resulting in at least 95 to 98% conversion.

9. A method for dual-fluorophore labeling of proteins for Forster Resonance Energy Transfer (FRET) assays, comprising:(a) performing the 3CR-2CP bioconjugation method as claimed in claim 1 to attach a first fluorophore at a designated carboxylate residue; and(b) performing a thiolyne or azide -alkyne cycloaddition reaction to attach a second fluorophore at a cysteine or other thiol-containing residue, wherein the first fluorophore is Cy3 while the second is Cy5, generating a C34- Cy5 / E45-Cy3 dual -labeled conjugate, the resulting dual-labelled protein exhibits measurable energy transfer efficiency (E FRET > 50 %).

10. The method as claimed in claim 1, wherein the synthesizing antibody- fluorophore conjugates (AFCs) or antibody-drug conjugates (ADCs) comprising performing the 4CR-2CP or 4CR-4CP reaction of as claimed in 1-9 on a monoclonal antibody to introduce orthogonal handles, followed by coupling with a fluorophore or cytotoxic drug, thereby yielding a homogeneous conjugate with antigen-specific binding and selective cytotoxicity.

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