FIELD OF INVENTION:

The invention is in the field of biotechnology and concerns protein labelling.

BACKGROUND OF THE INVENTION:

The Epsilon -Nth of lysine (Ν ^ε -ΝΗ ₂ ) is one of the most abundant naturally occurring reactive functional groups on the surface of proteins, among various other nucleophilic residues. Site- directed mutagenesis enabled the introduction of no n- natural amino acids into the protein backbone, and this has further enabled access to certain functional groups with unique reactivity. This specific non-proteinogenic residue is utilized for the attachment of a tag in subsequent bio-orthogonal transformation. For site-selective protein backbone modification (other than N-terminus) with endogenous/native proteins, proximity driven approach through ligand directed labeling is the only method. Chemical approaches were not in focus for site- selective protein labeling as it would involve addressing several challenging questions related to the selectivity. The pre -requisite of the labeling method involves the development of a chemical transformation that would be effective in near neutral aqueous buffer at ambient temperature. Besides, a functional group such as Ν ^ε -ΝΗ ₂ would have a stark confrontation with the other nucleophilic functionalities (chemoselectivity) and multiple Ν ^ε -ΝΗ ₂ residues (site- selectivity). The focal challenge revolves around the tenet that difference in nucleophilicity of multiple copies of an amino acid residue is inadequate to impart significant reactivity to a particular site. Thus, the technology still revolves around the most commonly adapted protocol that involves pre-engineered proteins with unnatural amino acids amenable to bio-orthogonal chemistry or enzyme-tag pairs. This limits the access of methodology to the majority of native proteins and antibodies. For example, if an affinity probe or fluorophore has to be attached to a native protein, it will result in a heterogeneously labeled mixture of proteins. Such limitation with the available methodology is also evident in the case of antibody drug conjugates (ADCs). The two approved drugs in this segment for directed therapeutics are synthesized using chemical approach. Both the drugs are available as heterogeneous mixtures. Another approach is the proximity driven approach and is driven by the specificity of ligand-receptor interactions. This technique has been applied for the site-specific modification of endogenous proteins. This chemo-enzymatic approach also has a limited set of applications. Hence, there is a need for an effective tool that would allow site-selective protein labeling of native (un-engineered) protein at single-site which can offer further flexibility to the user for the introduction of the desired tag in a late-stage modification.

OBJECT OF THE INVENTION:

The object of the invention is for a of method site-selective labeling of native (un-engineered) peptides and protein at single-site (Ν ^ε -ΝΗ ₂ ) which can offer further flexibility for installation of the desired tag in late-stage modification.

Another object of the invention is to synthesize labeled native proteins, labeled antibody-Fab fragment, and labeled monoclonal antibody, and for further synthesis of homogenous protein conjugates. FIGURES AND DRAWINGS:

Figure 1 depicts the reagents used in the method of the invention.

Figure 2 depicts reaction for the formylation and single site labelling of peptides. Figure 3 depicts reaction for the formylation and single site labelling of proteins. Figure 4 depicts reaction 1 and 2 for the formylation and interception of formylation for the site specific single site labelling of proteins.

Figure 5 depicts alkynylation of native proteins.

Figure 6 depicts aminophosphonation of native proteins. Figure 7 depicts tagging of a protein through (A) aldehyde component, and (B) late-stage oxime formation.

Figure 8 depicts aminophosphonation of antibody-Fab fragment and antibody for installation of drug molecule. DETAILED DESCRIPTION OF THE INVENTION:

Accordingly, the invention is for a method of site-selective single site labelling of peptides and proteins at single-site (Ν ^ε -ΝΗ2) which can offer further flexibility for installation of the desired tag in late-stage modification.

In addition, the invention is for the synthesis of labeled native proteins, labeled antibody-Fab fragment, and labeled monoclonal antibody and for further synthesis of homogenous protein conjugates.

Accordingly, chemical methods for the effective modification of single site labelling of proteins is described. In one embodiment, the functional groups introduced are the aldehyde, maleimide, and can be expanded to various bio-orthogonal functionalities. These functional groups enable the late- stage modification of the proteins.

The reagents involved in the introduction of the functional groups involve reagents such as aldehyde, alkyne, phosphite, and the like.

Labels can be introduced by using them as one of the components in reaction.

Labels introduced by late stage modification are affinity tags, biophysical probes such as fluorophore tags, and drug molecules.

The fluorophore tags is selected from 11a, lib, and 11c. It can be extended to hydroxylamine derivative of any fluorophore.

The drug molecules is selected from 12a and 12b. It can be extended to hydroxylamine derivative of any drug molecules.

The reaction time and temperature are with reference to the physiological conditions and can be varied depending on the molecules to be conjugated. The method of the introduction of the functional groups for single-site protein labeling are selected from (I) Formylation of protein (II) Interception of formylation (III) Alkynylation of protein (TV) Aminophosphonation of protein.

Further, by the method of the invention transformation results in Ν ^ε -formylation of lysine residue which is affected with the aldehyde as a formylating pre-reagent and which deliver high levels of efficiency with both peptides and native proteins in physiological conditions. Additionally, the method involves two sequential steps wherein the reversible reaction of the a-amine with aldehyde renders it unreactive towards further reaction with formate generated from an aldehyde or any electrophile to enable selective formylation of Ν ^ε -ΝΗ ₂ .

For the synthesis of proteins with the functional group with a bio-orthogonal site for late-stage modification of proteins with the desired tag, the Ν ^ε -formylation of lysine residue is intercepted by another electrophile, acylating reagent, to provide a site for late-stage modification. The interception of formylation can be performed by any electrophile delivered to the protein with a regulated rate of addition. The acylating reagents used for the introduction of the bio orthogonal functionalities is selected from 4a, 4b, 4c, 4d and 5.

In addition, for a single Ν ^ε -ΝΗ ₂ modification, chemoselective and site-selective ε-amine modification is performed with a multicomponent transformation that operates under physiological conditions in the presence of protein, aldehyde, acetylene, and Cu-ligand complex. The propargylamine is introduced at the labeled site is primed for late-stage bio- orthogonal modification.

Further, for a single Ν ^ε -ΝΗ ₂ modification, chemoselective and site-selective ε-amine modification is performed with a multicomponent transformation that operates under physiological conditions in the presence of protein, aldehyde, and triethylphosphite or diethylphosphite. Transformation is performed without the requirement of the metal catalyst. The aldehyde component derivatives can introduce tags directly or through a late-stage bio- orthogonal modification. Reagents used for technology I (-Formylation): la-g, 2a-c, 3a-n.

Reagents used for technology II (-Interception of formylation): la-g, 2a-c, 3a -n, 4a-d, 5.

Reagents used for technology III (-Alkynylation of protein): 3a, 6, 7a-g, 8a-b.

Reagents used for technology IV(-Aminophosphonation of protein.): la-g, 2a-c, 3a-n, 9, 10, lla-c.

Aldehyde reagents for the technologies I, II, III and IV: n = 1 -10, X = NH, O, S. R is independently selected from H; alkyl; cycloalkyl; aryl and Ri, R ₂ , R3 and R4 are independently selected from H; hydroxyl; -B(OR ^* )(O R ^** ) wherein R ^* and R ^** are independently selected from H; alkyl; lower alkyl; cycloalkyl; aryl; heteroaryl; alkenyl; heterocycle; halides; nitro; - C(0)OR ^* wherein R ^* is selected from H, alkyl; cycloalkyl and aryl; -C(0)NR ^** R ^*** , wherein R ^** and R ^*** are independently selected from H, alkyl; cycloalkyl and aryl; -CH2C(0)R _a , wherein R _a is selected from -OH, lower alkyl, cycloalkyl; aryl, -lower alkyl-aryl, -cycloalkyl- aryl; or -NRbR _c , where Rb and R _c are independently selected from H, lower alkyl, cycloalkyl; aryl or -lower alkyl-aryl; -C(0)Rd, wherein Rd is selected from lower alkyl, cycloalkyl; aryl or -lower alkyl-aryl; or -lower aikyl-OR _e , wherein R _e is a suitable protecting group or OH group. R5 , R6, and R7 are independently selected from H; nitro; cyano; halides; alkyl; cycloalkyl; aryl and C(0)OR ^* wherein R ^* is selected from H, alkyl; cycloalkyl and aryl; -C(0)NR ^** R ^*** , wherein R ^** and R ^*** are independently selected from H, alkyl; cycloalkyl and aryl. Rs and R9 are independently selected from H; halides; alkyl; cycloalkyl and aryl. Rio is selected from H; nitro; cyano; halides; alkyl; cycloalkyl; aryl and C(0)OR ^* wherein R ^* is selected from H, alkyl; cycloalkyl and aryl; -C(0)NR ^** R ^*** , wherein R ^** and R ^*** are independently selected from H, alkyl; cycloalkyl and aryl. Rn, R ₁₂ , R ₁ 3 and R14 are independently selected from H; alkyl; cycloalkyl; aryl and -SO3R wherein R is selected from H; Na. R15 and R½ are independently selected from H; alkyl; cycloalkyl; aryl and C(0)OR ^* wherein R ^* is selected from H, alkyl; cycloalkyl and aryl; -C(0)NR ^** R ^*** , wherein R ^** and R ^*** are independently selected from H, alkyl; cycloalkyl and aryl. R17 and Ri8 are independently selected from H; halides; alkyl; cycloalkyl and aryl. R19, R20, and R21 are independently selected from H; alkyl; aryl and C(0)OR ^* wherein R ^* is selected from H, alkyl; cycloalkyl and aryl; -C(0)NR ^** R ^*** , wherein R ^** and R ^*** are independently selected from H, alkyl; cycloalkyl and aryl.

The term "suitable substituent" is meant to include independently H; hydroxyl; cyano; alkyl, such as lower alkyl, such as methyl, ethyl, propyl, n-butyl, t-butyl, hexyl and the like; alkoxy, such as lower alkoxy such as methoxy, ethoxy, and the like; aryloxy, such as phenoxy and the like; vinyl; alkenyl, such as hexenyl and the like; alkynyl; formyl; haloalkyl, such as lower haloalkyl which includes CF3, CCI3 and the like; halide; aryl, such as phenyl and napthyl; heteroaryl, such as thienyl and furanyl and the like; amide such as C(0)NR**R***, , where R** and R*** are independently selected from lower alkyl, aryl or benzyl, and the like; acyl, such as C(0)-C6Hs, and the like; ester such as -C(0)OCH3 the like; ethers and thioethers, such as O-Bn and the like; thioalkoxy; phosphino; and -NRbRc, where Rb and R _c are independently selected from lower alkyl, aryl or benzyl, and the like. It is to be understood that a suitable substituent as used in the context of the present invention is meant to denote a substituent that does not interfere with the formation of the desired product by the processes of the present invention.

As used in the context of the present invention, the term "lower alkyl" as used herein either alone or in combination with another substituent means acyclic, straight or branched chain alkyl substituent containing from one to six carbons and includes for example, methyl, ethyl, 1 -methylethyl, 1 -methylpropyl, 2-methylpropyl, and the like. A similar use of the term is to be understood for "lower alkoxy", "lower thioalkyl", "lower alkenyl" and the like in respect of the number of carbon atoms. For example, "lower alkoxy" as used herein includes methoxy, ethoxy, i-butoxy.

The term "alkyl" encompasses lower alkyl, and also includes alkyl groups having more than six carbon atoms, such as, for example, acyclic, straight or branched chain alkyl substituents having seven to ten carbon atoms.

The term "aryl" as used herein, either alone or in combination with another substituent, means an aromatic monocyclic system or an aromatic polycyclic system. For example, the term "aryl" includes a phenyl or a napthyl ring, and may also include larger aromatic polycyclic systems, such as fluorescent (eg. anthracene) or radioactive labels and their derivatives.

The term "heteroaryl" as used herein, either alone or in combination with another substituent means a 5, 6, or 7-membered unsaturated heterocycle containing from one to 4 heteroatoms selected from nitrogen, oxygen, and sulphur and which form an aromatic system. The term "heteroaryl" also includes a polycyclic aromatic system comprising a 5, 6, or 7-membered unsaturated heterocycle containing from one to 4 heteroatoms selected from nitrogen, oxygen, and sulphur.

The term "cycloalkyl" as used herein, either alone or in combination with another substituent, means a cycloalkyl substituent that includes for example, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. The term also involves "cycloalkyl-alkyl-" that means an alkyl radical to which a cycloalkyl radical is directly linked; and includes, but is not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,

1 - cyclopentylethyl, 2-cyclopentylethyl, cyclohexylmethyl, 1 -cyclohexylethyl and

2- cyclohexylethyl. A similar use of the "alkyl" or "lower alkyl" terms is to be understood for aryl-alkyl-, aryl-lower alkyl- (eg. benzyl), -lower alkyl-alkenyl (eg. ally 1) , heteroaryl-alkyl-, and the like as used herein. For example, the term "aryl-alkyl-" means an alkyl radical, to which an aryl is bonded. Examples of aryl-alkyl- include, but are not limited to, benzyl (phenylmethyl), 1 -phenylethyl, 2-phenylethyl and phenylpropyl. As used herein, the term "heterocycle", either alone or in combination with another radical, means a monovalent radical derived by removal of a hydrogen from a three- to seven-membered saturated or unsaturated (including aromatic) heterocycle containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur. Examples of such heterocycles include, but are not limited to, pyrrolidine, tetrahydrofuran, thiazolidine, pyrrole, thiophene, hydantoin, diazepine, imidazole, isoxazole, thiazole, tetrazole, piperidine, piperazine, homopiperidine, homopiperazine, 1 ,4-dioxane, 4-morpholine, 4-thiomorpholine, pyridine, pyridine-N-oxide or pyrimidine, and the like.

The term "alkenyl", as used herein, either alone or in combination with another radical, is intended to mean an unsaturated, acyclic straight chain radical containing two or more carbon atoms, at least two of which are bonded to each other by a double bond. Examples of such radicals include, but are not limited to, ethenyl (vinyl), 1 -propenyl, 2-propenyl, and 1 -butenyl.

The term "alkynyl", as used herein is intended to mean an unsaturated, acyclic straight chain radical containing two or more carbon atoms, at least two of which are bonded to each other by a triple bond. Examples of such radicals include, but are not limited to, ethynyl, 1 -propynyl, 2-propynyl, and 1 -butynyl.

The term "alkoxy" as used herein, either alone or in combination with another radical, means the radical -0-(Ci- _n )alkyl wherein alkyl is as defined above containing 1 or more carbon atoms, and includes for example mefhoxy, ethoxy, propoxy, 1 -mefhylethoxy, butoxy and 1,1-dimethylethoxy. Where n is 1 to 6, the term "lower alkoxy" applies, as noted above, whereas the term "alkoxy" encompasses "lower alkoxy" as well as alkoxy groups where n is greater than 6 (for example, n = 7 to 10). The term "aryloxy" as used herein alone or in combination with another radical means -O-aryl, wherein aryl is defined as noted above. R5, R ₆ , and R7 can be sourced independently from any alkyl or cyclic alkyl functionalities that would not perturb the nucleophilicity of the phosphite.

R ₈ can be sourced from any alkyl or cyclic alkyl functionalities that would not perturb the nucleophilicity of hydroxylamine. For example, affinity tags, fluorescence tags, drugs, and toxins.

Further, several native proteins, antibody-Fab fragment, and a monoclonal antibody are synthesized with the functional groups by the site-selective labelling. Antibody drug conjugates were synthesized by using the doxorubicin derivative which acts as a fluorescent label and a drug.

EXAMPLES:

A more clear and concise functioning of the invention is provided in the examples and drawings, which are provided for better explanation and not intended to limit the disclosure of the invention.

EXAMPLE 1 :

(a) Formylation of protein

In a 2 ml Eppendorf tube, protein (14, 7.3 nmol) in phosphate buffer (95 μΐ, 0.1 M, pH 7.0) was taken. Aldehyde, ethyl-4-(4-formylphenoxy)butanoate (31, Ri, R2, R3, R4 = H, X = O and n = 2, 4.38 μmol ) in DMSO (5 μΐ) was taken from a freshly prepared stock solution and added to the protein followed by vortexing (350 rpm) at 25 °C or 37 °C. The overall concentration of protein (14a, 14c-14g) and aldehyde (31, R ₁ , R ₂ , R ₃ , R ₄ = H, X = O and n = 2) was 73 μΜ and 43.8 mM respectively. The progress of the transformation was followed by sampling aliquots at various time intervals by MALDI-ToF-MS using sinapic acid as matrix. After 24-48 h, the reaction mixture was further diluted with water (0.4 ml) and unreacted aldehyde was extracted using ethyl acetate/hexane (8:2, 5x1 ml). The reaction mixture was subjected to dialysis and the sample was concentrated by lyophilization before subjecting it to the digestion, peptide mapping and sequencing by MS-MS (Fig 1 and 2). In case of Melittin (14b), concentration of aldehyde (31, Ri, R ₂ , R3, R ₄ = H, X = O and n = 2, 4.38 μmol , 21.9 mM, 2.19 μmol ) was reduced to control the selectivity. All the other steps were similar to other proteins. In case of RNase A (14a) (7.3 nmol), 4-(decyloxy) benzaldehyde (3k, Ri, R ₂ , R3, R4 = H, X = O, R = C10H21, 4.38 μmol ) was used as formylating pre-reagent. The percent conversion and yield resulting out of the reaction is provided in Table 1.

Table 1 : % conversion and yield of the peptide/protein by the method

EXAMPLE 2:

(b) Interception of formylation

In a 2 ml Eppendorf tube, RNase A (14) (0 (7.3 nmol) in phosphate buffer (95 μΐ, 0.1 M, pH 7.0) was taken. Aldehyde, ethyl 4-(4-formylphenoxy) butanoate (31, Ri, R2, R3, R4 = H, X = O and n = 2, 4.38 μmol ) in DMSO (5 μΐ) was taken from a freshly prepared stock solution and added to the protein followed by vortexing (350 rpm) at 25 °C. After 3 h, 2,3,5,6- tetrafiuorophenyl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl) propanoate (4a, spacer la where n = 2, acylating reagent, 109.5 nmol) in DMSO (3 μΐ) from freshly prepared stock solution was added to the reaction mixture gradually over a period of 75 minutes. The concentration of the protein, aldehyde and acylating reagent (4a) in the reaction mixture was 73 μΜ, 43.8 mM and 1.09 mM respectively. The reaction was stopped in another 15 minutes by extraction of unreacted electrophile (acylating reagent) and aldehyde using ethyl acetate/he xane (8:2, 5x1 ml). The reaction mixture was subjected to dialysis and the sample was concentrated by lyophilization. Mono-labeled protein was dissolved in phosphate buffer (95 μΐ, 0.1 M, pH 7.0) and treated with 4-methyl-7-mercaptocoumarin (5, 3.65 mM, 365 nmole, from freshly prepared solution) in DMSO (5 μΐ) to give the fluorescent labeled protein. Protein labeling was monitored by using MALDI-ToF. After the late-stage modification, unreacted 4-methyl-7- mercaptocoumarin (5) was extracted using ethyl acetate/hexane (8:2, 5x1 ml). The reaction mixture was subjected to dialysis and the sample was concentrated by lyophilization, before subjecting it to digestion and peptide mapping. EXAMPLE 3:

(c) Alkynyhtion of protein (procedure 1 )

In a 1.5 ml Eppendorf tube, protein (14, 7.3 nmol) in phosphate buffer (90 μΐ, pH 7.8, 0.1 M) was taken. Formaldehyde (3a, R = H, 60 nl, 0.73 μmol ) in DMSO (3 μΐ) was taken from freshly prepared stock solution. To the reaction mixture, 1 , 10-phenanthroline (7g, 522 μg, 2.92 μmol ) in DMSO (2 μΐ), phenylacetylene (6, Ri, R ₂ , R ₃ , R4 = H, 78 nl, 0.73 μmol ) in DMSO (2.5 μΐ) and copper(I) iodide (8b, 120 μg, 0.73 μmol) in DMSO (2.5 μΐ) were added. After addition of all the reagents, a heterogeneous reaction mixture is formed that turns into a clear solution after 3-4 h. The reaction mixture was further vortexed for 72 h. Alkynylation of RNase A was followed by MALDI-ToF MS using sinapic acid as matrix. After 72 h, the reaction mixture was treated with hydroxylamine hydrochloride (250 μg, 3.65 μmol. ) in water (5 μΐ) for 6 h to remove the excess formaldehyde. The reaction mixture was subjected to dialysis [EDTA (0.05 M)-phosphate buffer (pH 7.8, 0.1 M)] and the sample was concentrated by lyophilization before subjecting it to digestion, peptide mapping, and sequencing by MS-MS.

(c) Alkynyhtion of protein (procedure 2 )

In a 0.5 ml Eppendorf tube, 1, 10-phenanthroline (7g, 522 μg, 2.92 μmol ) in DMSO (2 μΐ), phenylacetylene (6, Ri, R ₂ , R ₃ , R4 = H, 78 nl, 0.73 μmol ) in DMSO (2.5 μΐ) and copper(I) iodide (8b, 120 μg, 0.73 μmol ) in DMSO (2.5 μΐ) were mixed together and allowed to vortex for 1 h. In a separate 1.5 ml Eppendorf tube, protein (14, 7.3 nmol) in phosphate buffer (90 μΐ, pH 7.8, 0.1 M) and formaldehyde (3a, R = H, 60 nl, 0.73 μmol ) in DMSO (3 μΐ) were mixed. To the later mixture, pre-generated copper alkylidine complex was added. The reaction mixture is heterogeneous initially and turns into a clear solution after 3-4 h. The reaction mixture was further vortexed for 72 h. Alkynylation of protein was followed by MALDI-ToF MS using sinapic acid as matrix. After 72 h, the reaction mixture was treated with hydroxylamine hydrochloride (250 μg, 3.65 μmol ) in water (5 μΐ) for 6 h to remove the excess formaldehyde. The reaction mixture was subjected to dialysis [EDTA (0.05 M)-phosphate buffer (pH 7.8, 0.1 M)] and the sample was concentrated by lyophilization before subjecting it to digestion, peptide mapping and sequencing by MS-MS. The percent conversion and yield resulting out of the reaction is provided in Table 2.

Table 2: % conversion of the protein by the method

EXAMPLE 4:

(d) Aminophosphonation of protein

In a 1.5 ml Eppendorf tube, a protein (11, 7.3 nmol) in phosphate buffer (90 μΐ, pH 7.8, 0.1 M) was taken. Aldehyde (3b, R ₁ = Ph, 2.1 μιηοΐ) in DMSO (5 μΐ) and triethylphosphite (6, Rs, Re, R ₇ = Etthyl, 2.1 μιηοΐ) or diethylphosphite (7, R ₅ , R ₆ = Etthyl, 2.1 μιηοΐ) in DMSO (5 μΐ) were added from freshly prepared stock solutions. The reaction mixture was vortexed (350 rpm) in incubate shaker at 25 °C. The progress of reaction was monitored by MALDI-ToF MS using sinapic acid as matrix and ESI-MS. The excess aldehyde in the reaction mixture was quenched with hydroxylamine hydrochloride (250 μg, 3.65 μιηοΐ) in water (5 μΐ) for 2 h. The protein solution was desalted by using the concentrators (3 kDa MWCO) and then the sample was concentrated by lyophilization before subjecting it to digestion, peptide mapping and sequencing by MS-MS. The percent conversion and yield resulting out of the reaction is provided in Table 3.

Table 3: % conversion of the proteins by the method

ADVANTAGES:

The invention eliminates the requirement of pre-protein engineering and un-natural amino acids for labelling of proteins. Distinguishing a unique Lysine residue as a reactivity hotspot and open the doors for the single- site derivatization of proteins.

Application in probing protein interactions, ligand discovery, disease diagnosis, high- throughput screening, and regulating properties of therapeutic proteins. The later can be achieved by conjugating drugs, polymer chains, glycosylation, chromophores, and biohybrid materials to the proteins.