DESC:The following specification particularly describes the invention and the manner in which it is to be performed:
ACYLATION PROCESS FOR PREPARATION OF LIRAGLUTIDE

INTRODUCTION
Aspects of the present application relates to an acylation process for the preparation of Liraglutide.
Liraglutide, marketed under the brand name Victoza, is a long-acting glucagon like peptide agonist developed by Novo Nordisk for the treatment of type 2 diabetes.
Liraglutide is an injectable drug that reduces the level of sugar (glucose) in the blood. It is used for treating type 2 diabetes and is similar to exenatide (Byetta). Liraglutide belongs to a class of drugs called incretin mimetics because these drugs mimic the effects of incretins. Incretins, such as human-glucagon-like peptide-1 (GLP-1), are hormones that are produced and released into the blood by the intestine in response to food. GLP-1 increases the secretion of insulin from the pancreas, slows absorption of glucose from the gut, and reduces the action of glucagon. (Glucagon is a hormone that increases glucose production by the liver.) All three of these actions reduce levels of glucose in the blood. In addition, GLP-1 reduces appetite. Liraglutide is a synthetic (man-made) hormone that resembles and acts like GLP-1. In studies, Liraglutide treated patients achieved lower blood glucose levels and experienced weight loss.
Liraglutide is an acylated human glucagon-like peptide-1 (GLP-1) agonist, with a 97% amino acid sequence identity to endogenous human GLP-1(7-37). GLP-1(7-37) represents less than 20% of total circulating endogenous GLP-1. Like GLP-1(7-37), liraglutide activates the GLP-1 receptor, a membrane-bound cell-surface receptor coupled to adenylyl cyclase by the stimulatory G-protein, Gs, in pancreatic beta cells. Liraglutide increases intracellular cyclic AMP (cAMP), leading to insulin release in the presence of elevated glucose concentrations. This insulin secretion subsides as blood glucose concentrations decrease and approach euglycemia. Liraglutide also decreases glucagon secretion in a glucose-dependent manner. The mechanism of blood glucose lowering also involves a delay in gastric emptying. GLP-1(7-37) has a half-life of 1.5–2 minutes due to degradation by the ubiquitous endogenous enzymes, dipeptidyl peptidase IV (DPP-IV) and neutral endopeptidases (NEP). Unlike native GLP-1, liraglutide is stable against metabolic degradation by both peptidases and has a plasma half-life of 13 hours after subcutaneous administration. The pharmacokinetic profile of liraglutide, which makes it suitable for once daily administration, is a result of self-association that delays absorption, plasma protein binding and stability against metabolic degradation by DPP-IV and NEP. It reduces meal-related hyperglycemia (for 24 hours after administration) by increasing insulin secretion, delaying gastric emptying, and suppressing prandial glucagon secretion
Liraglutide, an analog of human GLP-1 acts as a GLP-1 receptor agonist. The peptide precursor of Liraglutide, produced by a process that includes expression of recombinant DNA in Saccharomyces cerevisiae, has been engineered to be 97% homologous to native human GLP-1 by substituting arginine for lysine at position 34. Liraglutide is made by attaching a C-16 fatty acid (palmitic acid) with a glutamic acid spacer on the remaining lysine residue at position 26 of the peptide precursor. The molecular formula of Liraglutide is C172H265N43O51 and the molecular weight is 3751.2 Daltons. It is represented by the structure of formula (I)

Formula (I)
U.S. Patent No. 7572884 discloses a process for preparing Liraglutide by recombinant technology followed by acylation and removal of N-terminal extension.
U.S. Patent No. 7273921 and 6451974 discloses a process for acylation of Arg-34GLP-1 to obtain Liraglutide.
WO98/08871 discloses acylation of GLP-1 and analogues.
WO98/08872 discloses acylation of GLP-2 analogues.
WO99/43708 discloses acylation of exendin and analogues.
Hence, there remains a need to provide alternate acylation process for the preparation of Liraglutide which is high yielding, scalable, cost effective, environment friendly and commercially viable by avoiding repeated cumbersome and lengthy purification steps.

SUMMARY
In the first embodiment, the present invention provides a process for the preparation of N-substituted peptide or protein comprising:
(a) reacting a peptide or protein with a copper agent to form a copper complex of peptide or protein,
(b) converting the copper complex of the peptide or protein obtained in step (a) to the desired N-substituted peptide or protein.
In the second embodiment, the present invention provides a process for the preparation of N-acylated peptide or protein comprising:
(a) reacting a peptide or protein with a copper agent to form a copper complex of peptide or protein,
(b) converting the copper complex of peptide or protein obtained in step (a) to the desired N-acylated peptide or protein.
In the third embodiment, the present invention provides a process for the preparation of N-substituted peptide or protein comprising:
(a) reacting a peptide or protein with a copper agent to form a copper complex of peptide or protein,
(b) converting the copper complex of peptide or protein obtained in step (a) to a N-substituted peptide or protein.
(c) hydrolyzing the N-substituted peptide or protein obtained in step (b) to obtain the desired N-substituted peptide or protein.
In the fourth embodiment, the present invention provides a process for the preparation of N-acylated peptide or protein comprising:
(a) reacting a peptide or protein with a copper agent to form a copper complex of peptide or protein,
(b) converting the copper complex of peptide or protein obtained in step (a) to a N-acylated peptide or protein.
(c) hydrolyzing the N-acylated peptide or protein obtained in step (b) to obtain the desired N-acylated peptide or protein.
In the fifth embodiment, the present invention provides a process for the preparation of N-substituted GLP analogues comprising:
(a) reacting a GLP analogues with a copper agent to form a copper complex of GLP analogues,
(b) converting the copper complex of GLP analogues obtained in step (a) to the desired N-substituted GLP analogues.
In the sixth embodiment, the present invention provides a process for the preparation of N-acylated GLP analogues comprising:
(a) reacting a GLP analogues with a copper agent to form a copper complex of GLP analogues,
(b) converting the copper complex of GLP analogues obtained in step (a) to the desired N-acylated GLP analogues.
In the seventh embodiment, the present invention provides a process for the preparation of N- substituted GLP analogues comprising:
(a) reacting a GLP analogues with a copper agent to form a copper complex of GLP analogues,
(b) converting the copper complex of GLP analogues obtained in step (a) to N-substituted GLP analogues.
(c) hydrolyzing the N-substituted GLP analogues obtained in step (b) to obtain the desired N-substituted GLP analogues.
In the eighth embodiment, the present invention provides a process for the preparation of N-acylated GLP analogues comprising:
(a) reacting a GLP analogues with a copper agent to form a copper complex of GLP analogues,
(b) converting the copper complex of GLP analogues obtained in step (a) to an N-acylated GLP analogues.
(c) hydrolyzing the N-acylated GLP analogues obtained in step (b) to obtain the desired N-acylated GLP analogues.
In the ninth embodiment, the present invention provides a process for the preparation of liraglutide comprising:
(a) reacting liraglutide precursor with a copper agent to form a copper complex of liraglutide precursor,
(b) converting the copper complex of liraglutide precursor obtained in step (a) to liraglutide.
In the tenth embodiment, the present invention provides a process for the preparation of liraglutide comprising:
(a) reacting a liraglutide precursor with a copper agent to form a copper complex of liraglutide precursor,
(b) converting the copper complex of liraglutide precursor obtained in step (a) to N-substituted liraglutide precursor.
(c) hydrolyzing the N-substituted liraglutide precursor obtained in step (b) to obtain liraglutide.

DETAILED DESCRIPTION
The present invention involves formation of copper complex of a peptide or a protein with a copper agent. Copper (II) can form stable complexes with amino acids, peptides or proteins through chelation. A copper agent as used herein comprises copper salts such as cupric hydroxide, copper carbonate, copper chloride, copper sulfate and the like.
Suitable solvent that can be used for the formation of copper complex includes water; C1-C10 straight or branched chain alcohol such as methanol, ethanol, isopropyl alcohol, 1-butanol, 2-butanol, 2-methyl-2-propanol, 1-pentanol, 2-pentanol, 2,2-dimethyl-1-propanol, 2,2,2-trimethyl ethanol, 1-decanol, benzyl alcohol; nitriles such as acetonitrile, propionitrile; ethers such as tetrahydrofuran, dioxane, diisopropylether, diethylether, dibutyl ether, 2-methyltetrahydrofuran, cyclopentyl methyl ether or methyl tert-butyl ether; esters such as ethyl formate, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, methyl propanoate, ethyl propanoate, methyl butanoate or ethyl butanoate; ketones such as acetone, butanone, pentanone, methyl isobutyl ketone; halogenated solvents such as dichloromethane, chloroform, tetrachloromethane, dichloroethane, chlorobenzene or dichlorobenzene; aliphatic hydrocarbon solvents such as methylcyclohexane, cyclohexane, heptane or hexane; aromatic hydrocarbon solvents such as toluene, benzene, chlorobenzene, 4-chlorotoluene, trifluorotoluene, o-xylene, m-xylene or p-xylene; polar aprotic solvents such as N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, pyridine, dimethylsulphoxide, sulpholane, formamide, acetamide, propanamide; or mixtures thereof.
The reaction time for the copper complex formation should be sufficient to complete the reaction which depends on scale and mixing procedures, as is commonly known to one skilled in the art. Typically, the reaction time can vary from about few minutes to several hours. For example the reaction time can be from about 10 minutes to about 24 hours, or any other suitable time period.
Suitable temperatures for the copper complex formation may be less than 120°C, less than 100°C, less than 80°C, less than 60°C, less than 40°C, less than 20°C, less than 10°C, or any other suitable temperatures.
The copper complex formation described herein can be carried out in air or under an inert atmosphere. Typically, reactions containing reagents or products that are substantially reactive with air can be carried out under inert atmosphere such as nitrogen, argon, helium or the like.
In the present invention, conversion of the copper complex of the peptide or protein to the desired N-substituted peptide or protein or to the desired N-acylated peptide or protein can be carried out in the presence or absence of solvent. In one variant, the solvent is a non-aqueous organic solvent. In another variant, the solvent can be as described herein above.
The reaction time for the conversion of the copper complex of the peptide or protein to the desired N-substituted peptide or protein or to the desired N-acylated peptide or protein should be sufficient to complete the reaction which depends on scale and mixing procedures, as is commonly known to one skilled in the art. Typically, the reaction time can vary from about few minutes to several hours. For example the reaction time can be from about 10 minutes to about 24 hours. In one variant, it can be about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 5 hours, about 8 hours, about 10 hours, about 15 hours, about 20 hours, about 24 hours or any other suitable time period.
Suitable temperatures for the conversion of the copper complex of the peptide or protein to the desired N-substituted peptide or protein or to the desired N-acylated peptide or protein may be less than 120°C, less than 100°C, less than 80°C, less than 60°C, less than 40°C, less than 20°C, less than 10°C, or any other suitable temperatures.
The conversion of the copper complex of the peptide or protein to the desired N-substituted peptide or protein or to the desired N-acylated peptide or protein described herein can be carried out in air or under an inert atmosphere. Typically, reactions containing reagents or products that are substantially reactive with air can be carried out under inert atmosphere such as nitrogen, argon, helium or the like.
In a preferred embodiment, N-acylation is done by employing compound of Formula I.

n is 0-8; R1 is COOR4; R2 is a lipophilic moiety; R3 and its attached carboxyl group designate a reactive ester or a reactive N-hydroxy imide ester; and R4 is selected from the group consisting of hydrogen, C1-12 alkyl and benzyl.
In certain embodiments when R4 is not hydrogen, the process of the present invention further involves hydrolysis of N-substituted/N-acylated peptide or protein to the desired N-substituted/N-acylated peptide or a protein.
N-substituted peptide or protein can be any N-substituted peptide or protein that has desired N-substitution including N-acyl, N-alkyl or the like.
Optionally, the N-substituted peptide or protein obtained in step (b) of the embodiments of the present invention may contain functional groups which have to be subjected to a functional group transformation to obtain the desired N-substituted peptide or protein.
In one variant, the said N-substituted peptide or protein obtained in step (b) may contain functional groups such as esters, O-alkyl derivatives such as C1-12-alkyl, e.g. methyl, ethyl, prop-1-yl, prop-2-yl, but-1-yl, but-2-yl, 2-methyl-prop-1-yl, 2-methyl-prop-2-yl (tert-butyl), hex-1-yl, etc., aryl derivatives like benzyl, alkyloxy or aryloxy derivatives.
When the said functional group is a carboxylic acid ester; such ester can be hydrolyzed by basic hydrolysis or acidic hydrolysis to obtain the desired N- substituted peptide or protein containing the free carboxylic acid group.
Basic hydrolysis can be carried out using bases such as alkali metal hydroxides including sodium hydroxide, potassium hydroxide, lithium hydroxide or the like; alkali metal carbonates including sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate or the like; ammonia, sodium t-butoxide, potassium t-butoxide, sodium methoxide, and the like.
The acidic hydrolysis can be carried out using inorganic or organic acids, but are not carboxylic acids. Suitable inorganic acids are those having pKa values below about 4.0 at room temperature in aqueous solution (see Moeller, Inorganic Chemistry, John Wiley & Sons (1952) at pages 314 and 315). Specific examples of such acids are sulfuric acid which is a preferred strong acid catalyst and hydrochloric acid, perchloric acid, nitric acid, phosphoric acid, and hydrofluoric acid. Organic acids suitable for strong acid catalysts herein are noncarboxylic acids having pKa values below 2.0 in water at room temperature (see Handbook of Chemistry and Physics, 58th edition, Chemical Rubber Publishing Company at pages D-150 et seq.). Examples of suitable organic acids are methane sulfonic acid, naphthalene sulfonic acid, trifluoromethyl sulfonic acid, and p-toluene sulfonic acid. Mixtures of strong acid catalysts can also be used.
Suitable solvent that can be used for the said ester hydrolysis includes water; C1-C10 straight or branched chain alcohol such as methanol, ethanol, isopropyl alcohol, 1-butanol, 2-butanol, 2-methyl-2-propanol, 1-pentanol, 2-pentanol, 2,2-dimethyl-1-propanol, 2,2,2-trimethyl ethanol, 1-decanol, benzyl alcohol; nitriles, such as acetonitrile, propionitrile; ethers such as tetrahydrofuran, dioxane, diisopropylether, diethylether, dibutyl ether, 2-methyltetrahydrofuran, cyclopentyl methyl ether or methyl tert-butyl ether; esters such as ethyl formate, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, methyl propanoate, ethyl propanoate, methyl butanoate or ethyl butanoate; ketones such as acetone, butanone, pentanone, methyl isobutyl ketone; halogenated solvents such as dichloromethane, chloroform, tetrachloromethane, dichloroethane, chlorobenzene or dichlorobenzene; aliphatic hydrocarbon solvents such as methylcyclohexane, cyclohexane, heptane or hexane; aromatic hydrocarbon solvents such as toluene, benzene, chlorobenzene, 4-chlorotoluene, trifluorotoluene, o-xylene, m-xylene or p-xylene; polar aprotic solvents, such as for example, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, pyridine, dimethylsulphoxide, sulpholane, formamide, acetamide, propanamide; or mixtures thereof.
The reaction time for ester hydrolysis should be sufficient to complete the reaction which depends on scale and mixing procedures, as is commonly known to one skilled in the art. Typically, the reaction time can vary from about few minutes to several hours. For example the reaction time can be from about 10 minutes to about 24 hours, or any other suitable time period.
Suitable temperatures for the said hydrolysis may be less than 120°C, less than 100°C, less than 80°C, less than 60°C, less than 40°C, less than 20°C, less than 10°C, or any other suitable temperatures.
Optionally the desired N-substituted peptide or protein can be isolated by techniques known in the art. For example, isolation can be done by removal of solvent from the solution containing the product. Suitable techniques which can be used for the removal of solvent include but not limited to evaporation, flash evaporation, simple evaporation, rotational drying, spray drying, agitated thin-film drying, agitated nutsche filter drying, pressure nutsche filter drying, freeze-drying or any other technique known in the art.
Optionally, in carrying out the processes according to the present invention, the reaction product of a given step can be carried forward to the next step without the isolation of the product from the previous step i.e., one or more reactions in a given process can be carried out in-situ as one pot process optionally in the presence of the same reagent/s used in a previous step wherever appropriate to do so, to make the process of the present invention economical and commercially more viable.
The resulting compounds may be optionally further dried. Drying can be carried out in a tray dryer, vacuum oven, air oven, cone vacuum dryer, rotary vacuum dryer, fluidized bed dryer, spin flash dryer, flash dryer, lyophilizer, or the like. The drying can be carried out at temperatures of less than about 60°C, less than about 50°C, less than about 40°C, less than about 30°C, less than about 20°C, or any other suitable temperatures; at atmospheric pressure or under a reduced pressure; as long as the Liraglutide is not degraded in its quality. The drying can be carried out for any desired time until the required product quality is achieved. Suitable time for drying can vary from few minutes to several hours for example from about 30 minutes to about 24 or more hours.
Optionally, in carrying out the processes according to the present invention, the reaction product of a given step can be isolated and purified by the methods described herein or the methods known to a person skilled in the art before using in a subsequent step of the process.
Purification processes include but not limited to preparative reverse phase HPLC, ion exchange chromatography, size exclusion chromatography, affinity chromatography or any other well-known technique in the art.
Room temperature as used herein refers to ‘the temperatures of the thing close to or same as that of the space, e.g., the room or fume hood, in which the thing is located’. Typically, room temperature can be from about 20°C to about 30°C, or about 22°C to about 27°C, or about 25°C.
The reactions of the processes described herein can be carried out in air or under an inert atmosphere. Typically, reactions containing reagents or products that are substantially reactive with air can be carried out using air-sensitive synthetic techniques that are well known to the person skilled in art.

DEFINITIONS
The following definitions can be used in connection with the words or phrases used in the present application unless the context indicates otherwise.
The term "peptide" as used herein refers to any peptide comprising two or more amino acid residues connected by peptide linkage.
The term “protein” as used here in refers to large molecule composed of one or more chains of amino acids in a specific order.
The term “peptide or a protein” as used here in refers to GLP analogues or any other peptide or protein, which contain two or more terminal and/or side chain amino groups.
The term “GLP analogues” as used herein refers to GLP-1, GLP-2, and GLP derivatives such as GLP-1(1-35), GLP-1 (1-36), GLP-1(1-36)amide, GLP-1(1-37), GLP-1(1-38), GLP-1(1-39), GLP-1(1-40), GLP-1(1-41) or an analogue thereof. Preferred GLP derivatives include but not limited to Arg26-GLP-1(1-37); Arg34-GLP-1(7-37); Arg34Lys26-GLP-1(7-37); Lys36-GLP-1-(7-37); Arg26,34Lys36-GLP-1(7-37); Arg26,34Lys38GLP-1(7-38); Arg26,34Lys39-GLP-1(7-39); Arg26,34Lys40-GLP-1(7-40); Arg26Lys36GLP-1(7-37); Arg34Lys36-GLP-1(7-37); Arg26Lys39-GLP-1(7-39); Arg34Lys40GLP-1(7-40); Arg26,34Lys36,39-GLP-1(7-39); Arg26,34Lys36,40-GLP-1(7-40); Gly8Arg26-GLP-1(7-37); Gly8Arg34-GLP-1(7-37); Gly8Lys36-GLP-1(7-37); Gly8Arg26,34Lys36GLP-1(7-37); Gly8Arg26,34Lys39-GLP-1(7-39); Gly8Arg26,34Lys40-GLP-1(7-40); Gly8Arg26Lys36GLP-1(7-37); Gly8Arg34Lys36-GLP-1(7-37); Gly8Arg26Lys39-GLP-1(7-39); Gly8Arg34Lys40GLP-1(7-40); Gly8Arg26,34Lys36,39-GLP-1(7-39); or Gly8Arg26,34Lys36,40-GLP-1(7-40).
The term “N-substituted peptide or protein” as used herein refers to Arg34Lys26-[N-e-(?-Glu(N-hexadecanoyl))]-a-methyl-GLP-1(7-37), Arg34Lys26-[N-e-(?-Glu(N-hexadecanoyl))]-a-ethyl-GLP-1(7-37), Arg34Lys26-[N-e-(?-Glu(N-hexadecanoyl))]-a-butyl-GLP-1(7-37), Arg34Lys26-[N-e-(?-Glu(N-hexadecanoyl))]-a-benzyl-GLP-1(7-37), Glu22,23,30Arg26,34Lys38(Ne-(?-glutamyl(Na-tetradecanoyl)))-a-methyl-GLP-1(7-38)-OH, Glu23,26Arg34Lys38(Ne-(?-glutamyl(Na-tetradecanoyl)))- a-methyl -GLP-1(7-38)-OH, Lys26,34-bis(Ne-(?-glutamyl(Na-tetradecanoyl)))-a-methyl-GLP-1(7-37)-OH, Lys26,34-bis(Ne-(?-glutamyl(Na-hexadecanoyl)))-a-methyl-GLP-1(7-37)-OH, or the like.
The term “N-acylated peptide or protein” as used herein refers to Arg34Lys26-[N-e-(?-Glu(N-hexadecanoyl))]-a-methyl-GLP-1(7-37), Arg34Lys26-[N-e-(?-Glu(N-hexadecanoyl))]-a-ethyl-GLP-1(7-37), Arg34Lys26-[N-e-(?-Glu(N-hexadecanoyl))]-a-butyl-GLP-1(7-37), Arg34Lys26-[N-e-(?-Glu(N-hexadecanoyl))]-a-benzyl-GLP-1(7-37), Glu22,23,30Arg26,34Lys38(Ne-(?-glutamyl(Na-tetradecanoyl)))-a-methyl-GLP-1(7-38)-OH, Glu23,26Arg34Lys38(Ne-(?-glutamyl(Na-tetradecanoyl)))- a-methyl -GLP-1(7-38)-OH, Lys26,34-bis(Ne-(?-glutamyl(Na-tetradecanoyl)))-a-methyl-GLP-1(7-37)-OH, Lys26,34-bis(Ne-(?-glutamyl(Na-hexadecanoyl)))-a-methyl-GLP-1(7-37)-OH, or the like.
The term “desired N-substituted peptide or protein” as used herein refers to Liraglutide, Lys26(Ne-tetradecanoyl)-GLP-1(7-37), Lys34(Ne-tetradecanoyl)-GLP-1(7-37), Arg26,34Lys36 (Ne-tetradecanoyl)-GLP-1(7-37)-OH, Lys26,34bis(Ne-(?-carboxynonadecanoyl))-GLP-1(7-37)-OH, Arg26,34Lys38(Ne-(?-carboxynonadecanoyl))-GLP-1(7-38)-OH, Arg34Lys26 (Ne-(?-carboxynonadecanoyl))-GLP-1(7-37)-OH, Arg26,34Lys38(Ne-(?-carboxyundecanoyl))-GLP-1(7-38)-OH, Arg34Lys26 (Ne-(?-carboxyundecanoyl))-GLP-1(7-37)-OH, Arg34Lys26(Ne-lithocholyl)-GLP-1(7-37)-OH, Glu22,23,30Arg26,34Lys38(Ne-(?-glutamyl(Na-tetradecanoyl)))-GLP-1(7-38)-OH, Glu23,26Arg34Lys38(Ne-(?-glutamyl(Na-tetradecanoyl)))-GLP-1(7-38)-OH, Lys26,34-bis(Ne-(?-glutamyl(Na-tetradecanoyl)))-GLP-1(7-37)-OH, Lys26,34-bis(Ne-(?-glutamyl(Na-hexadecanoyl)))-GLP-1(7-37)-OH, Arg26,34Lys38(Ne-(?-glutamyl(Na-tetradecanoyl)))-GLP-1(7-38)-OH or the like.
The term “desired N-acylated peptide or protein” includes N-acylated GLP analogues such as Liraglutide, Lys26(Ne-tetradecanoyl)-GLP-1(7-37), Lys34(Ne-tetradecanoyl)-GLP-1(7-37), Arg26,34Lys36 (Ne-tetradecanoyl)-GLP-1(7-37)-OH, Lys26,34bis(Ne-(?-carboxynonadecanoyl))-GLP-1(7-37)-OH, Arg26,34Lys38(Ne-(?-carboxynonadecanoyl))-GLP-1(7-38)-OH, Arg34Lys26 (Ne-(?-carboxynonadecanoyl))-GLP-1(7-37)-OH, Arg26,34Lys38(Ne-(?-carboxyundecanoyl))-GLP-1(7-38)-OH, Arg34Lys26 (Ne-(?-carboxyundecanoyl) )-GLP-1(7-37)-OH, Arg34Lys26(Ne-lithocholyl)-GLP-1(7-37)-OH, Glu22,23,30Arg26,34Lys38(Ne-(?-glutamyl(Na-tetradecanoyl)))-GLP-1(7-38)-OH, Glu23,26Arg34Lys38(Ne-(?-glutamyl(Na-tetradecanoyl)))-GLP-1(7-38)-OH, Lys26,34-bis(Ne-(?-glutamyl(Na-tetradecanoyl)))-GLP-1(7-37)-OH, Lys26,34-bis(Ne-(?-glutamyl(Na-hexadecanoyl)))-GLP-1(7-37)-OH, Arg26,34Lys38(Ne-(?-glutamyl(Na-tetradecanoyl)))-GLP-1(7-38)-OH or any other like thereof.
The term "amino acid" as used herein refers to an organic compound comprising at least one amino group and at least one acidic group. The amino acid may be a naturally occurring amino acid or be of synthetic origin, or an amino acid derivative or amino acid analog.
The term “acylating” as used herein refers to the introduction of one or more acyl groups covalently bonded to the free amino groups of the peptide or protein.
The term “acylation” means the acylation of the amino group of the peptide or a protein.
Although the exemplified procedures herein illustrate the practice of the present invention in some of its embodiments, the procedures should not be construed as limiting the scope of the invention. Modifications from consideration of the specification and examples within the ambit of current scientific knowledge will be apparent to one skilled in the art.

EXAMPLES
Example 1: Preparation of 5-t-butyl-1-methyl palmityl glutamate.
In a 250 mL dry bottom flask, palmitic acid (3.0 g, 11.70 mmol) was dissolved in 50mL of dichloromethane at room temperature under argon atmosphere. Triethylamine (3.3mL, 23.64 mmol) was then added drop wise and the mixture was stirred for 5 min. 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uranium hexafluorophosphate (HATU) (6.74 g, 17.7 mmol) was added and allowed to stir for additional 10 min. In a separate round bottom flask, L-Glutamic acid tert-butyl ester (3.0g, 11.82) was taken in 30ml of dichloromethane and then added dropwise to the above activated palmitic acid in dichloromethane solution. The reaction mixture was allowed to stir at room temperature overnight. The suspension was filtered, quenched with water, extracted with dichloromethane and dried using sodium sulphate. It is then purified by silica gel column chromatography using methanol and dichloromethane to yield 5-t-butyl-1-methyl palmityl glutamate as a white solid. (5.37g, 99.7% yield). ESI-MS (+ve) m/Z: 456.63(MH+).
Example 2: Synthesis of 1-methyl palmityl glutamic acid.
To a solution of 5-t-butyl-1-methyl palmityl glutamate (5.37g, 11.78 mmol) in dichloromethane (30mL), trifluoroacetic acid (9mL, 118mmol) was added and allowed to stir at room temperature for 24h. The resulting solution was quenched with water and extracted using dichloromethane. The solvents were removed under reduced pressure to give 1-methyl palmityl glutamic acid as a dry white solid (4.82g, 99%) ESI-MS (+ve) m/Z: 400.62(MH+).
Example 3: Synthesis of 1-methyl palmityl glutamic acid.
To a solution of 5-t-butyl-1-methyl palmityl glutamate (82 g, 147 mmol) in dichloromethane (450mL), trifluoroacetic acid (139mL, 1800mmol) were added and allowed to stir at room temperature for 24h. The resulting solution was quenched with water and extracted using dichloromethane. The solvents were removed under reduced pressure to give 1-methyl palmityl glutamic acid as a dry white solid.

Example 4: Synthesis of 5-(2, 5-dioxopyrrolidin-1-yl) 1-methyl palmitoyl-L-glutamate
To a solution of 1-methyl palmityl glutamic acid (74.5 g, 186 mmol) in tetrahydrofuran (630 mL), diisopropylcarbodiimide (DIC) (29 mL, 186 mmol) was added at room temperature and stirred for 15 min. Then, N-hydroxy succinimide (21.4 g, 186 mmol) was added to the above mixture and allowed to stir overnight at room temperature. The suspension was quenched with water (500 mL) and extracted with dichloromethane (2000 mL) and dried over sodium sulphate. It was then purified using silica gel chromatography using hexane and dichloromethane to afford the title compound as dry white solid.
Example 5: Synthesis of 5-(2, 5-dioxopyrrolidin-1-yl) 1-methyl palmitoyl-L-glutamate
To a solution of 1-methyl palmityl glutamic acid (202mg,0.5mmol)in tetrahydrofuran (5 mL) was added 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (98 mg, 0.51mmol) at room temperature and stirred for 15 min. N-hydroxy succinimide (58.9 mg, 0.51mmol) and triethyl amine (111µl, 1.01 mmol)were added to the above solution and allowed to stir overnight at room temperature. The suspension was filtered and the filtrate was evaporated to dryness, quenched with water and extracted with dichloromethane and dried over sodium sulphate. It was then purified using silica gel chromatography using methanol and dichloromethane. ESI-MS (+ve) m/Z: 497.52(MH+).
Example 6: Conjugation of lirapeptide with palmityl glutamate derivatives.
1.2 M CuSO4 5H2O solution (5 µl, 5.9 µmol) was added directly to the lirapeptide (20mg, 5.9 µmol) and azeotropically dried using toluene and dissolved in dimethylformamide (1mL) at room temperature. Triethylamine was added for a period of 10min. and the suspension was stirred for 5 min. Tert.butyl dimethyl silyl chloride (4.5 mg, 29.5 µmol) was added and stirred for another 10 min. In a separate flask, 1-methyl palmityl glutamic acid (2.38 mg, 5.96 µmol) in dimethyl formamide (1ml) was activated using 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uranium hexafluorophosphate (HATU) (2.26 mg, 5.96 µmol) and 2,6-lutidine (1.4 µl, 11.92 µmol) were added drop wise to the above reaction mixture. Additional 6 equivalents of 2, 6-lutidine (4.1 µl, 35.7 µmol) and 2 equivalents of activated 1-methyl palmityl glutamic acid were added for the reaction to complete. The progress of the reaction was monitored by UPLC (86% conversion based on lirapeptide area) and the yield of the reaction was estimated to contain 51% (based on area) by analytical RP-HPLC.
Example 7: Conjugation of lirapeptide with N-hydroxy succinimide ester of palmitoyl glutamic acid in an organic medium.
To a solution of lirapeptide (10mg, 2.95 µmol) in dimethylformamide (1 mL) was added 0.8 µl of triethyl amine (0.8 µl, 5.9 µmol) at room temperature and stirred for 5 min. 1.2 M CuSO4 5H2O solution (2.4 µl, 5.9 µmol) was added and the mixture stirred for 10 min. Tert.butyl dimethyl silyl chloride (2.2 mg, 14.74 µmol) was added and stirred for 10 min. 0.08 M solution of N-hexadecanoyl glutamic acid ?-N-hydroxysuccinimide ester in N-methylpyrrolidone (37 µl, 2.95 µmol) and 2, 6-lutidine (2.4 µl, 20.65 µmol) were added and stirred at room temperature. Additional 2 equivalents of N-hexadecanoyl glutamic acid ?-N-hydroxysuccinimide ester were added for the reaction to complete. The progress of the reaction was monitored by UPLC (96.8% conversion based on lirapeptide area) and the yield of the reaction was estimated to contain 72.5% (based on area) by analytical RP-HPLC.
Example 8: Conjugation of lirapeptide with N-hydroxy succinimide ester of palmitoyl glutamic acid in an aqueous medium.
To a solution of lirapeptide (5.08mg, 1.5 µmol) in water (1 mL) at 0°C was added 1.6 µl of triethyl amine (0.84 µl, 6 µmol) and 1.2 M CuSO4 5H2O solution (1.3 µl, 1.5 µmol) and stirred for 5 min. 0.08 M solution of N-hexadecanoyl glutamic acid ?-N-hydroxysuccinimide ester in N-methylpyrrolidone (24 µl, 1.95 µmol) was added and stirred at 0°C. Then 0.12M solution of NaHCO3 (480 µl, 6 µmol) and 0.08M solution of N-hexadecanoyl glutamic acid ?-N-hydroxysuccinimide ester in N-methylpyrrolidone (24 µl, 1.95 µmol) were added at 0°C and slowly warmed to room temperature. The progress of the reaction was monitored by UPLC (90% conversion based on lirapeptide area) and the yield of the reaction was estimated to contain 66.7% (based on area) by analytical RP-HPLC.
Example 9: Conjugation of lirapeptide with N-hydroxy succinimide ester of palmitoyl glutamic acid in an aqueous medium.
To a solution of lirapeptide (2 g, 0.59 mmol) in water (400 mL) at 0°C, triethyl amine (40 µl, 0.27 mmol) and 1.2 M CuSO4 5H2O solution (493 µl, 0.59 mmol) were added and stirred for 5 min. Then, additional triethylamine (83 µl, 0.6 mmol) was added to the above reaction mixture in order to maintain the pH ~9.5. Then, 0.08 M solution of N-hexadecanoyl glutamic acid ?-N-hydroxysuccinimide ester in N-methylpyrrolidone (7.5 mL, 0.59 mmol) was added at 0°C and mixture was slowly warmed to room temperature and stirred overnight. Additional, triethylamine (20 µl, 0.14 mmol) and N-hexadecanoyl glutamic acid ?-N-hydroxysuccinimide ester (440 mg) in N-methylpyrrolidone (11mL) was added for progress of reaction. The progress of the reaction was monitored by UPLC (93% conversion based on lirapeptide area).
Example 10: Conjugation of lirapeptide with N-hydroxy succinimide ester of palmitoyl glutamic acid in an aqueous medium.
To a solution of lirapeptide (8.6 g, 2.54 mmol) in water (2800 mL), triethyl amine (2 mL,14.3 mmol) and 0.2 M CuSO4 5H2O solution (0.634 g, 2.54 mmol) were added at at 0°C and stirred for 5 min. Then, additional triethylamine (0.400mL, 2.86 mmol) was added to the above reaction mixture in order to maintain the pH ~10.0. Then, 420 mL of acetonitrile was added and mixture was stirred for 5 minutes. Then 0.01 M solution of N-hexadecanoyl glutamic acid ?-N-hydroxysuccinimide ester in acetonitrile (698 mL, 6.98 mmol) was added at 0°C over a period of 1.5 hours and mixture was slowly warmed to room temperature and stirred for 2-3 hours till the reaction was >95% complete.
Example 11: Hydrolysis of Liraglutide methyl ester to obtain liraglutide
The crude reaction mixture containing Liraglutide methyl ester (10mg, 2.9 µmol) in dimethylformamide was evaporated to dryness and then dissolved in water, pH was adjusted using 1M NaOH and stirred at room temperature for 1h. Progress of the reaction was monitored by UPLC which showed the complete conversion of Liraglutide methyl ester to Liraglutide.
Example 12: Hydrolysis of Liraglutide methyl ester to obtain liraglutide
To the crude reaction mixture of Liraglutide methyl ester (2.2 g, 0.59 mmol) in water (435 mL), sodium hydroxide solution (5N, 2.5mL, 12.5mmol) was added and the resulting solution was stirred at room temperature for 3 hours. Progress of the reaction was monitored by UPLC which showed the complete conversion of Liraglutide methyl ester to Liraglutide.

Example 13: Hydrolysis of Liraglutide methyl ester to obtain liraglutide
To the crude reaction mixture of Liraglutide methyl ester (9.6 g, 2.54 mmol) in water:acetonitrile mixture (3918mL, 30% acetonitrile in water), aqueous solution of lithium hydroxide monohydrate (3g in 75 mL of water) was added and the resulting solution was stirred at pH ~ 12.8 at room temperature for 2 hours. Progress of the reaction was monitored by UPLC. After completion of the reaction, pH was adjusted to ~ 8.5 using 5.2 mL of trifluoroacetic acid and then 0.5 M EDTA (7.62 mL, pH = 8.5) was added and then stirred at room temperature for 2hours. Then acetonitrile was evaporated under vacuum and the crude Liraglutide product was obtained by precipitation at the isoelectric point of 4.7 using 5.3mL of trifluoroacetic acid and 1 mL of 0.5N sodium hydroxide.
Example 14: Purification of Liraglutide using High Pressure Reverse Phase Purification System
Liraglutide crude product (14.2 grams of wet pellet) was dissolved in 100mM Tris pH 8.5±0.1 (913 ml) at a concentration of 1.95g/L. This crude mixture was centrifuged at 8500 rpm, 20 min, 23±2 °C, to remove particulate matter. The supernatant was filtered through 1.2 µm followed 0.8 µm and 0.45µm PES filters. The resin used for separation is Reverse phase kromasil/Dasiogel 10 µm C8 100 Å (Dimensions 50x250mm, 491ml CV) which had been equilibrated with (10% of mobile phase B (Mobile Phase A: 10 mm Tris pH 8.5±0.1; Mobile phase B: 100% Acetonitrile) at the linear flow rate of 230cm/hr before loading. The amount of product loaded onto the resin is 4.36 grams of product/litre of resin or 0.73% gram of product /gram of resin. The column was washed with 3 CV (Column Volume) of 10% mobile phase B and then with 3 CV of 25% of mobile phase B at a linear flow rate of 230cm/hr. The Liraglutide was eluted with 15 CVs of 28-42 % of mobile phase B and during elution multiple fractions (23ml) were collected. The Liraglutide in each fraction was addressed for purity by analytical HPLC method. The peak fractions whose purity was greater than 98% were combined to make Elution pool (380ml). Typically acetonitrile present in Elution pool was 31 to 34% (v/v) and was evaporated using rotary evaporator at 22 °C, pI precipitated using acetic acid (100µL) and centrifuged at 8500 rpm for 20 min at 5±2°C. Finally, the pellet was lyophilized at 0.16mbar for 24hours and 1.08 grams of 98.46% pure white powder obtained.
,CLAIMS:WE CLAIM:
Claim 1: A process for the preparation of N-substituted peptide or protein comprising,
a) reacting a peptide or protein with a Copper agent to form a copper complex of peptide or protein,
b) converting the copper complex of the peptide or protein obtained in step (a) to the desired N-substituted peptide or protein.
c) optionally, hydrolyzing the N-substituted peptide or protein obtained in step (b) to obtain the desired N-substituted peptide or protein.

Claim 2: The process of claim 1, wherein copper agent in step a) comprises of copper salts such as cupric hydroxide, copper carbonate, copper chloride, copper sulfate and the like.

Claim 3: The process of claim 2, wherein copper sulfate pentahydrate is employed in step a).

Claim 4: The process of claim 1, wherein step b) involves reaction with a suitable compound to afford the desired N-substituted peptide or protein.

Claim 5: The process of claim 4, wherein N-substituted peptide or protein is Liraglutide.

Claim 6: The process of claim 5, wherein the N-acylating agent comprises of 1-methyl palmityl glutamic acid or its reactive derivatives.

Claim 7: The process of claim 4, wherein step b) is performed in an organic solvent or in water or mixtures thereof.

Claim 8: The process of claim 1 comprising an additional step of hydrolyzing the N-substituted peptide or protein obtained in step (b) to obtain the desired N-substituted peptide or protein.

Claim 9: The process of claim 8, wherein the N-substituted peptide or protein in step (b) is Liraglutide alkyl ester.

Claim 10: The process of claim 8, wherein the desired N-substituted peptide or protein is Liraglutide.