JP5551670B2 - Peptide acylation method and novel acylating agent - Google Patents

Description

  The present invention relates to a method for introducing one or more acyl groups into a peptide or protein. More particularly, the present invention relates to an improved acylation method for the ε-amino group of lysine residues contained in natural GLP-1 or analogs thereof. Furthermore, the present invention relates to a compound effective as an acylating agent in the above method.

  Since peptides are widely used in the medical field and can be produced by recombinant DNA technology, their importance is expected to continue to increase. When natural peptides or their analogs are used for therapy, it is widely seen that they have high clearance. The high clearance of therapeutic agents is inconvenient if it is desired to maintain their high blood concentration over an extended period of time even after repeated administration is required. Examples of naturally occurring peptides with high clearance are: ACTH, corticotropin releasing factor, angiotensin, calcitonin, exendin, exendin-3, exendin-4, insulin, glucagon, glucagon-like peptide-1, glucagon-like peptide-2 , Insulin-like growth factor-1, insulin-like growth factor-2, gastric inhibitory peptide, growth hormone releasing factor, pituitary adenylate cyclase activating peptide, secretin, enterogastrin, somatostatin, somatotropin, somatomedin, parathyroid gland Hormone, thrombopoietin, erythropoietin, hypothalamic hormone releasing factor, prolactin, thyroid stimulating hormone, endorphin, enkephalin, vasopressin, oxytocin, opi Id and their analogs, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase, and ribonuclease.

  Introduction of a lipophilic acyl group into a natural peptide or analog thereof leads to an acylated peptide with prolonged action characteristics associated with the natural peptide (or an unmodified analog). This phenomenon can be attributed to the applicant's earlier application, WO 98/08871, which discloses acylation of GLP-1 and analogs, WO 98/08872, which discloses acylation of GLP-2 and analogs, and exendin and analog acylation. Fully described and demonstrated in the disclosed WO 99/43708. Furthermore, it has been proposed that the inclusion of groups which may have a negative charge adjacent to the lipophilic group, for example carboxylic acid groups, may be advantageous.

  European patent application 921070335.5 (Kuraray) describes a reactive monoester of a long chain dicarboxylic acid for the preparation of long chain carboxylic acids to proteins.

  The introduction of lipophilic acyl groups into GLP-1 via mono- or dipeptide spacers is particularly interesting and has been proposed and exemplified in WO 98/08871. Aspartic acid and glutamic acid have been mentioned as suitable linkers. However, since such mono- or dipeptide spacers contain additional carboxylic acid groups, it was thought that protection and subsequent deprotection steps were necessary. Deprotection was performed under acidic conditions that led to a certain degree of degradation of the peptide (GLP-1). Thus, alternative methods for the preparation of these variants are desirable.

  Thus, it is an object of the present invention to provide an alternative method for the introduction of lipophilic residues into peptides via an α-amino-α, ω-dicarboxylic acid spacer. Said method will facilitate the preparation of modified peptides. Carboxylic acid groups which can be charged are introduced in the vicinity of the lipophilic residue and do not act directly on the lipophilic residue.

The present invention is a method for acylating one or more amino groups of a peptide (or protein) to obtain an N-acylated peptide (N-acylated protein) comprising the following steps:
(A) A peptide (or protein) having at least a free amino group is represented by the following general formula (I):

{Where,
n is 0-8;
R ¹ is COOR ⁴ ;
R ² is a lipophilic moiety, for example selected from C _3-39 -alkyl, C _3-39 -alkenyl, C _{3-39 -alkadienyl} , and steroidal residues;
R ³ together with the carboxyl group to which R ³ is attached represents a reactive ester or a reactive N-hydroxy imide ester; and R ⁴ is hydrogen, C _1-12 -alkyl And benzyl. }
And (b) when R ⁴ is not hydrogen, the above acylated peptide, under basic conditions, with an acylating agent represented by Saponifies the ester group (COOR ⁴ );
A method comprising the steps of:

Saponification of the acylated peptide ester (where R ⁴ is an alkyl group or benzyl group) under basic conditions may result in very little racemization or non-racemization of the various α-amino acid fragments of the peptide and the spacer. Possible in racemization. Certain advantages have been discovered in this application with respect to previously used acidic hydrolysis related to the purity and inhibition of by-products such as degradation products.

Acylation with an acylating agent as the free acid (here R ⁴ is hydrogen) under basic conditions has also been discovered. Said acylation directly leads to the desired product, the acylated peptide, essentially without side products and without a deprotection step.

  The present invention also provides a novel compound effective as an acylating agent in the above-mentioned method, wherein the novel compound is represented by the following general formula (I):

{Where,
n is 0-8;
R ¹ is COOH;
R ² is a lipophilic moiety, eg selected from C _3-39 -alkyl, C _3-39 -alkenyl, C _{3-39 -alkadienyl} , and steroidal residues; and R ³ is attached to R ³ Reactive ester or reactive N-hydroxy imide ester is represented together with the carboxyl group. }
It is represented by

Peptides and proteins It is generally understood that the present invention is effective for the introduction of lipophilic acyl groups into some peptides (or proteins) to reduce the clearance rate in vivo. Examples of said peptides and proteins are ACTH, corticotropin releasing factor, angiotensin, calcitonin, exendin and its analogs, insulin and its analogs, glucagon and its analogs, glucagon-like peptide-1 and its analogs, glucagon-like peptide-2 and Its analogs, insulin-like growth factor-1, insulin-like growth factor-2, gastric inhibitory peptide, growth hormone releasing factor, pituitary adenylate cyclase activating peptide, secretin, enterogastrin, somatostatin, somatotropin, somatomedin, Parathyroid hormone, thrombopoietin, erythropoietin, hypothalamic hormone releasing factor, prolactin, thyroid stimulating hormone, endorphin, enkephalin, batho Lessin, oxytocin, opioids and analogues thereof, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase, and ribonuclease.

  It will be appreciated that the peptide (or protein) will have at least one free amino group, wherein the amino group will have an N-terminal amino group or a side chain amino group. Of particular interest are the amino groups of lysine and ornithine amino acid residues. Said method relates in particular to N-acetylation of the ε-amino group of lysine residues. It will also be understood that the peptide or protein of interest may have more than one pendant amino group that can be N-acylated in accordance with the present invention.

  It will be appreciated that the present invention is particularly suitable for modification of GLP-1 and its analogs. Examples of GLP-1 and analogs that can be N-acylated in accordance with the present invention include GLP-1 and truncated analogs such as

It is.

  Each of these GLP-1 analogs and truncated analogs constitutes another aspect of the invention.

It will be appreciated that the present invention is also particularly suitable for modification of GLP-2 and its analogs. Examples of GLP-2 and analogs that can be N-acylated in accordance with the present invention include GLP-2 analogs and truncated analogs such as Lys ²⁰ GLP-2 (1-33); Lys ²⁰ Arg ³⁰ GLP-2 (1-33). ^{); Arg 30 Lys 34 GLP-} 2 (1-34); Arg 30 Lys 35 GLP-2 (1-35); Arg 30,35 Lys 20 GLP-2 (1-35); and ^Arg 35 GLP-2 ( 1-35). Each of these GLP-2 analogs and truncated analogs constitutes another aspect of the invention.

  It will be understood that the present invention is also particularly suitable for modification of exendin and its analogs. Examples of exendins and analogs that can be N-acylated in accordance with the present invention are exendin analogs and truncated analogs such as exendin-3 and exendin-4. Each of these exendin analogs and truncated analogs constitutes another aspect of the present invention.

  In a further embodiment of the invention said N-acylation occurs at the ε-amino group of the lysine residue.

  The actions of GLP-1 and its analogs are all described in WO 98/08871.

  The actions of GLP-2 and its analogues are all described in WO 98/08872.

  The actions of exendin and its analogs are all described in WO 99/43708.

Acylating Agent In the method of the present invention, a peptide (or protein) having at least one free amino group is reacted with an acylating agent of general formula (I).

  The integer n in the general formula (I) is preferably 0 to 8, particularly 0 to 6, and corresponds to, for example, aspartic acid or glutamic acid. Preferably n is 0-4, for example 0-2, 0 (aspartic acid) or 1 (glutamic acid). Each of these integers and ranges constitutes another aspect of the invention.

R ¹ in formula (I) represents a free acidic group (COOH) or an ester group (COOR ⁴ ). In the case where R ¹ is an ester group, R ⁴ is selected from a group that can be liberated by hydrolysis (as corresponding to an alcohol) under basic conditions. Examples of said groups are C _1-12 -alkyl, such as 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 and the like and benzyl. Each of these groups constitutes another aspect of the invention.

R ² in formula (I) represents a lipophilic moiety incorporated in the peptide or protein. Said lipophilic moiety is typically selected from _C3-39 -alkyl, _C3-39 -alkenyl, _C3-39 -alkadienyl, and steroidal residues. Specific examples of C _3-39 -alkyl are heptyl, nonyl, undecanyl, tridecanyl, pentadecanyl, heptadecanyl, and nonadecanyl. Each of these lipophilic portions constitutes another aspect of the invention.

  Said lipophilic substituent or moiety is for water at 20 ° C. in the range of about 0.1 mg / 100 ml of water to about 250 mg / 100 ml of water, preferably in the range of about 0.3 mg / 100 ml of water to about 75 mg / 100 ml of water. It is characterized by the solubility it has. For example, octanoic acid (C8) has a solubility of 68 mg / 100 ml in 20 ° C. water, decanoic acid (C10) has a solubility of 15 mg / 100 ml in 20 ° C. water, and octadecanoic acid (C18) Has a solubility of 0.3 mg / 100 ml in 20 ° C. water. Each of these lipophilic substituent ranges constitutes another aspect of the invention.

The term _{"C 3-39} - alkyl _(C 3-39 _{-alkyl)", "C 3-39} - alkenyl _(C 3-39 _-alkenyl)", and _{"C 3-39} - alkadienyl _{(C 3-39} - alkadienyl) is intended to include straight and branched chains, preferably straight chain, saturated, mono-unsaturated, di-unsaturated, each of hydrocarbon radicals of 3 to 39 carbon atoms. Specific examples of C _3-39 -alkyl are heptyl, nonyl, undecanyl, tridecanyl, pentadecanyl, heptadecanyl, and nonadecanyl.

As used herein, the term “steroidal residues” is intended to mean a lipophilic group with a carbonyl group to which R ² derived from a steroidal carboxylic acid is attached. For example, tri-, tetra-, and _pentacyclic , fully saturated or partially unsaturated C _{16-36 -hydrocarbons} . Examples of said group R ² —C (═O) — are lithocoloyl, deoxycholoyl, and coroyl.

Of the lipophilic groups, C _7-25 -alkyl, C _7-25 -alkenyl, C _7-25 -alkadienyl, and steroidal residues listed above are particularly relevant. Particularly interesting examples are heptyl, nonyl, undecanyl, tridecanyl, pentadecanyl, heptadecanyl, nonadecanyl, lithocoyl, deoxycoroyl, and coroyl. Each of these lipophilic groups constitutes another aspect of the invention.

R ³ in formula (I) represents a reactive ester or a reactive N-hydroxy imide ester together with a carboxyl group to which R ³ is attached. Each of these esters constitutes another aspect of the invention. Reactive esters or reactive N-hydroxyimide esters are well known in the field of organic chemistry (especially peptide chemistry) as reactive groups used for acylation of amino, thio, and hydroxy groups. The term “reactive ester or reactive N-hydroxyimide ester” in the context of the present invention means an ester having a functionality suitable for acylation of an amine, preferably a primary amine, in the form of a carboxylic acid group. Intended to be. Thus, it will be appreciated that the selectivity of acylation of primary amines preferably extends to the acylation of hydroxy and thio groups. Reactive N-hydroxy imide esters are particularly preferred.

  Examples of reactive esters are 1-hydroxybenzotriazole esters and derivatives. Many highly effective reagents for the formation of said activated esters of carboxylic acids, for example 2- (1H-benzotriazol-1yl) -1,1,3,3-tetramethylronium tetrafluoroborate It has been known. The reactive ester is typically formed in situ by the presence of a base, for example a trialkylamine, which is an organic base.

  Examples of the imide part of N-hydroxy imide ester are those clearly described from page 13 line 3 to page 17 line 10 of European Patent Application No. 921070335.5. Incorporated herein by reference). Among them, examples of particularly interesting imide moieties include succinimide, phthalimide, and the like. Each of these imide moieties constitutes another aspect of the present invention.

The reactive N-hydroxy imide ester of formula (I) has the corresponding acid (ie, N-acylated α-carboxy protected diacid (R ⁴ is N-acylated α-carboxy protected diacid) Not hydrogen)) and equimolar amounts (eg 0.95 to 1.05 moles, preferably 1.0 moles) of the corresponding imides may be prepared by condensation of N-hydroxy-imides. (The N-acylated α-carboxy protected diacid is typically prepared from the corresponding lipophilic moiety of the α-carboxy protected α-aminodiaside, and a benzotriazole ester. This benzotriazole ester is prepared, for example, from chloric acid and benzotriazole or from free acid and benzotriazole by DCC conjugation as described in WO 98/02460 (Examples 1-3). For example, in the presence of a conjugating agent, such as a carbodiimide conjugating agent (eg, dicyclohexylcarbodiimide (DCC)). In this case, the conjugate is preferably added in an equimolar amount with respect to the acid. The reaction is typical in polar aprotic solvents such as anhydrous tetrahydrofuran (THF), anhydrous dimethylformamide (DMF), anhydrous acetone, anhydrous dichloromethane, anhydrous dioxane, anhydrous dimethylacetamide or anhydrous N-methyl-2-pyrrolidone (NMP). Is done. The reaction is typically carried out at a temperature in the range of 0-50 ° C., for example 5-30 ° C., for example room temperature, for a period of 1-96 hours, for example 4-36 hours. One possible combination of reagents and conditions is the following: anhydrous THF or anhydrous DMF (or mixtures thereof) in an approximate 1: 1 molar ratio of the N-hydroxy-imide (succinimide or phthalimide) to the acid of interest. Dissolve in and add an equimolar amount of DCC to the solution. After completion of the reaction between N-hydroxy-imide and acid, the product is purified by conventional methods such as filtration (filtration of precipitated dicyclohexylurea (DCU) if DCC is used as a conjugating agent), crystallization, recrystallization. Separation and purification by crystallization, chromatography, etc. One feasible purification route is filtration, evaporation of the solvent under reduced pressure, redissolution of the product, e.g. redissolved in acetone, filtration, crystallization by addition of a non-polar solvent, e.g. redissolved in hexane, and in some cases Removal of used conjugating agents precipitated by recrystallization and / or washing. Said product can be used immediately in the process of the invention as said acylating agent of formula (I).

When the acylating agent of formula (I) is used as a free alpha carboxylic acid (R ⁴ = hydrogen), the compound of formula (I) above, wherein R ⁴ is a group that is selectively removed, is R Convert to the corresponding compound where ⁴ is hydrogen. The carboxylic acid protecting group can be a benzyl group that can be removed by catalytic hydrogenation or an allyl group that can be selectively removed. Benzyl protecting groups can be removed by catalytic hydrogenation in an aprotic polar solvent, for example palladium on carbon and hydrogen at room temperature in acetone. The reaction can also take place in a closed vessel (typically 0.1-10 atm) in a hydrogen gas atmosphere under vigorous stirring. The reaction is typically complete in 0.5 to 12 hours depending on the quality of the palladium catalyst. Apply conventional methods.

It will be understood that the compounds of the above formula (I) in which R ⁴ is hydrogen are novel substances, whereby they constitute a special aspect of the present invention. Thus, the present invention provides the following general formula (I):

{Where,
n is 0-8;
R ¹ is COOH;
R ² is a lipophilic moiety and is preferably selected from C _3-39 -alkyl, C _3-39 -alkenyl, C _{3-39 -alkadienyl} , and steroidal residues; and R ³ is R ³ Together with the carboxyl group attached to it, represents a reactive ester or a reactive N-hydroxy imide ester. }
A novel compound represented by is also provided.

Reaction conditions The reaction between the acylating agent of formula (I) and the peptide or protein is carried out under basic conditions in a mixture of an aprotic polar solvent and water.

  The acylating agent of formula (I) is typically used in a slight excess relative to the number of amino groups in the peptide or protein. The ratio is typically 1: 1 to 1:20 and extra acylating agent, preferably 1: 1.2 to 1: 5, taking into account the value of the number of amino groups in the peptide.

  It will be understood that whether the peptide is fully N-acylated or only partially N-acylated can depend on the amount of acylating agent used and the reaction conditions. It is preferred that the N-acylation is substantially stoichiometric.

  Typically, the aprotic polar solvent is from anhydrous tetrahydrofuran (THF), anhydrous dimethylformamide (DMF), acetone, dichloromethane, dimethyl sulfoxide (DMSO), dioxane, dimethylacetamide or N-methyl-2-pyrrolidone and mixtures thereof. Among them, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, and N-methyl-2-pyrrolidone are preferred, and N-methyl-2-pyrrolidone is particularly preferred. The ratio of said aprotic polar solvent to water (eg N-methyl-2-pyrrolidone and water) is typically 1:10 to 10: 1, especially 1: 5 to 5: 1, especially 1: 1 to 3: 1.

  The temperature is typically maintained in the range of -10 to 50 ° C, preferably in the range of 0 to 25 ° C.

  In order for the reaction to proceed smoothly, it is important that the pH value of the solvent mixture is in the range of 7 to 14, for example 9 to 13, preferably 9.5 to 12.5. Results regarding yield and purity are generally optimal when the pH value of the solvent mixture is in the range of 10-12. The desired pH value is obtained by addition of alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, and / or organic bases such as trialkylamines (triethylamine, N, N-diisopropylethylamine, etc.).

  As a typical example, the reaction of step (a) is carried out by use of the protein and the acylating agent of formula (I) in a molar ratio of 1: 1 to 1: 5. The peptides are typically pre-dissolved in water at -10-30 ° C, eg 0-25 ° C, and the pH adjusted to the desired level using an alkali metal hydroxide (eg sodium hydroxide or potassium hydroxide). Has been. The pH value can be further adjusted using acids, such as acetic acid and bases, such as trialkylamines, but the temperature is preferably within the above range. The aprotic polar solvent (or solvent mixture) is then added. The reaction is typically performed by condensation (observed by HPLC) in 0.2 to 4 hours, for example 0.2 to 1 hour, prior to addition of water and acid, for example acetic acid up to pH 6.5-9.0. ) Typically proceed. The products are typically separated and purified by precipitation by HPLC or isoelectric point pH or by hydrolysis prior to purification (step (b)).

When an acylating agent of the above formula (I) wherein R ⁴ is hydrogen is used, an N-acylated peptide or a protein with a lipophilic moiety and a free carboxylic acid group is obtained directly. Thus, variants in which R ⁴ is hydrogen represent a preferred embodiment of the method of the present invention.

Alternatively, ie when R ⁴ is C _1-12 -alkyl or benzyl, the N-acylated peptide ester (or protein ester) is N-acylated peptide or N-acylated. Is saponified under basic conditions to obtain a new protein. Saponification is typically performed in a 0.01-4.0 M solution of an alkali metal hydroxide, such as sodium or potassium hydroxide. The pH of the solution is typically 10-14. The reaction is typically allowed to proceed for 0.1-12 hours, preferably 0.5-4 hours, 0-40 ° C., for example at about room temperature. After the reaction, the product is purified by, for example, precipitation at isoelectric point pH and / or preliminary HPLC. Thus, variants in which R ⁴ is C _1-12 -alkyl or benzyl represent another preferred embodiment of the method of the present invention.

The invention also relates to the following aspects:
Side 1. A method for acylating an amino group of a peptide or protein to obtain an N-acylated peptide comprising the following steps:
(A) A peptide having at least one free amino group is represented by the following general formula (I):

{Where,
n is 0-8;
R ¹ is COOR ⁴ ;
R ² is a lipophilic moiety;
R ³ together with the carboxyl group to which R ³ is attached represents a reactive ester or a reactive N-hydroxy imide ester; and R ⁴ is hydrogen, C _1-12 -alkyl And benzyl. }
Reacting in a mixture of an aprotic polar solvent and water under basic conditions with an acylating agent represented by: and (b) when R ⁴ is not hydrogen, the above acylated peptide under basic conditions Saponifying the ester group (COOR ⁴ );
Including said method.

Side 2. The method of aspect 1, wherein R ⁴ is hydrogen.

Side 3. The process of aspect 1, wherein R ⁴ is selected from C _1-8 -alkyl and benzyl.

Side 4. Wherein R ³ together with the carboxyl group of where R ³ is attached thereto, represent a reactive N- hydroxy imide ester method according to any of claims 1 to 3.

  Side surface 5. 5. The method according to any of aspects 1 to 4, wherein the mixture of aprotic polar solvent and water is a mixture of N-methyl-2-pyrrolidone and water up to 1: 5 to 5: 1.

  Side 6. The method according to any one of aspects 1 to 5, wherein the pH of the reaction mixture in step (a) is in the range of 9 to 13.

  Side surface 7. The method according to any one of Sides 1 to 6, wherein the temperature of the reaction mixture in step (a) is in the range of 0 to 50 ° C.

  Side surface 8. 8. A method according to any of aspects 3 to 7, wherein the acylated peptide ester is saponified at a pH value in the range of 10-14.

Side 9. R ² is _{C 3-39} - _{alkyl, C 3-39} - _{alkenyl, C 3-39} - alkadienyl, and are selected from steroidal residues A method according to any of aspects 1-8.

  Side 10. The following formula (I):

{Where,
n is 0-8;
R ¹ is COOH;
R ² is a lipophilic moiety; and R ³ together with the carboxyl group to which R ³ is attached represents a reactive ester or a reactive N-hydroxy imide ester. }
A compound represented by

Side 11. 11. A compound according to aspect 10, wherein n is 0 or 1 and R ³ represents a reactive ester or a reactive N-hydroxy imide ester together with a carboxyl group to which R ³ is attached.

Side 12. R ² is _{C 3-39} - _{alkyl, C 3-39} - _{alkenyl, C 3-39} - alkadienyl, and are selected from steroidal residues, the compounds according to aspect 10 or 11.

Preparation of starting material Example 1 Preparation of N-hexadecanoyl glutamic acid α-benzyl ester Glutamic acid α-benzyl ester (4.75 g, 20.0 mmol) in N-methyl-2-pyrrolidone (100 ml) 20-25 Dissolved at ° C. 1-Hexadecanoylbenzotriazole (7.15 g, 20.0 mmol) was added simultaneously with triethylamine (2.53 g, 25.0 mmol). The reaction mixture was stirred at 20-25 ° C. for 22 hours. 0.2M hydrochloric acid (250 ml) was added to the product solution. The product suspension was cooled at 0 ° C. for 3 hours. The product was separated by filtration, washed with water (50 ml × 4) and dried to constant weight under reduced pressure at 40 ° C.

  Yield: 9.15 g (96%) of white material, melting at 90.0 ° C. (peak value) was measured by differential thermal analyzer (DSC).

Example 2 Preparation of N-hexadecanoyl glutamic acid α-methyl ester Using 8.06 g (50.0 mmol) of glutamic acid α-methyl ester under reaction conditions similar to those described in Example 1, N- Hexadecanoyl glutamic acid α-methyl ester was prepared.

  Yield: 17.70 g (88%) of white material, melting at 95.4 ° C. (peak value) was measured by DSC.

Example 3 Preparation of N-hexadecanoyl glutamic acid α-benzyl ester γ-N-hydroxysuccinimide ester N-hexadecanoyl glutamic acid α-benzyl ester (23.78 g, 50.0 mmol) was added to tetrahydrofuran (200 ml). Was dissolved at 20-25 ° C. Dicyclohexylcarbodiimide (10.32 g, 50.0 mmol) was added simultaneously with N-hydroxysuccinimide (5.75 g, 50.0 mmol). The reaction mixture was stirred at 20-25 ° C. for 20 hours. The product suspension was filtered and the filtrate was evaporated by drying under reduced pressure. The crystalline residue was dissolved in acetone (100 ml) at 40 ° C. and impurities were removed by filtration. N-Heptane (300 ml) was added to the filtrate. The product suspension was stirred at 20-25 ° C. for 4 hours and then cooled at 0 ° C. for 1/2 hour. The product was separated by filtration, washed with n-heptane (50 ml × 3) and dried to constant weight under reduced pressure at 40 ° C.

  Yield: 23.75 g (83%) of white material, melting at 98.6 ° C. (peak value) was measured by DSC.

Example 4 Preparation of N-hexadecanoyl glutamic acid α-methyl ester γ-N-hydroxysuccinimide ester 8.00 g (20 mmol) of N-hexadecanoyl glutamic acid under reaction conditions similar to those described in Example 3 N-hexadecanoyl glutamic acid α-methyl ester γ-N-hydroxysuccinimide ester was prepared using α-methyl ester.

  Yield: 6.45 g (65%) of white material, melting at 106.0 ° C. (peak value) was measured by DSC.

Example 5 Preparation of N-hexadecanoyl glutamic acid γ-N-hydroxysuccinimide ester N-hexadecanoyl glutamic acid α-benzyl ester γ-N-hydroxysuccinimide ester (5.73 g, 10.0 mmol) was prepared. Dissolved in acetone (100 ml) at 20-25 ° C. Carbon paste containing 10% palladium (about 0.25 g as a dry product) was added. The suspension was stirred under a hydrogen atmosphere until no more hydrogen was consumed (290 ml of hydrogen, 45 minutes). The catalyst was removed by filtration and the filtrate was evaporated by drying under reduced pressure at 20-25 ° C. The residue was dissolved in acetone (25 ml) at 20-25 ° C. and impurities were removed by filtration. N-Heptane (200 ml) was added to the filtrate. The product suspension was stirred at 20-25 ° C. for 1 hour. The product was separated by filtration, washed with n-heptane (50 ml × 2) and dried to constant weight under reduced pressure at 40 ° C.

  Yield: 4.20 g (87%) of white material, melting at 100.8 ° C. (peak value) was measured by DSC.

Preparation Example of acylated GLP-1 analogue ^{6 Arg 34 Lys 26 - [N} -ε- (γ-Glu (N- hexadecanoyl))] - Preparation of ^{GLP-1 7-37} Arg ³⁴ -GLP- 1 ^7-37 (freezing material 5.0g of precipitated isoelectric point peptide, about 0.15 mmol) was dissolved at 0 to 5 ° C. water (25 ml). The pH of the solution was adjusted to 12.5 by adding 1.0 M sodium hydroxide (2.25 ml). Two minutes later, N-methyl-2-pyrrolidone (50 ml) and 1.0 M acetic acid (1.25 ml) were added while keeping the temperature at 15 ° C. Triethylamine (0.2 ml) and N-hexadecanoyl glutamic acid γ-N-hydroxysuccinimide ester (97.0 mg, 0.20 mmol) were added simultaneously. After 30 minutes, 15 ° C. water (50 ml) was added and the pH was adjusted to 8.0 by addition of 1.0 M acetic acid (1.70 ml).

Yield: said reaction mixture by RP-HPLC for analysis ^{Arg 34 Lys 26 - [N-} ε- (γ-Glu (N- hexadecanoyl))] - a ^{GLP-1 7-37} (in area) 77% containing Showed that to do.

  Final purification of the product was performed by column chromatography.

Example ^{7 Arg 34 Lys 26 - [N} -ε- (γ-Glu-OMe (N- ^{hexadecanoyl))] - GLP-1 7-37} N in the reaction conditions similar to that described in Preparation Example 6 -Hexadecanoylglutamic acid α-methyl ester γ-N-hydroxysuccinimide ester was used to acylate Arg ³⁴ -LP-1 ^7-37 .

Yield: said reaction mixture by RP-HPLC for analysis ^{Arg 34 Lys 26 - [N-} ε- (γ-Glu-OMe (N- hexadecanoyl))] - a ^{GLP-1 7-37} (in area) 64 % Content. The product could be isolated as a precipitate by adjusting the pH of the reaction mixture to 6.0 using 1M acetic acid. Alternatively, the reaction mixture can be used immediately as described in Example 8 below.

Example ^{8 Arg 34 Lys 26 - [N} -ε- (γ-Glu (N- hexadecanoyl))] - The reaction mixture containing the product obtained in Preparation Example 7 of ^{GLP-1 7-37,} 1M It was subjected to basic hydrolysis by adjusting the pH to 12-13 with sodium hydroxide. The temperature of the reaction mixture was maintained at 8-18 ° C. After 2 hours, the reaction was complete and the pH of the reaction mixture was adjusted to 7.45 by addition of 1M acetic acid.

Yield: said reaction mixture by RP-HPLC for analysis ^{Arg 34 Lys 26 - [N-} ε- (γ-Glu (N- hexadecanoyl))] - a ^{GLP-1 7-37} (in area) containing 65% Showed that to do.

  Final purification of the product was performed by column chromatography.

Example 9
below:
^{Arg 26,34, Lys 36 - [N} -ε- (γ-Glu (N- ^{hexadecanoyl))] - GLP-1 7-36} , Arg 26, Lys 34 - [N-ε- (γ-Glu ( N- hexadecanoyl))] - ^{GLP-1 7-37,} and ^{Gly 8, Arg 26,34, Glu 37} , Lys 38 - [N-ε- (γ-Glu (N- hexadecanoyl))] - GLP-1 ^7-38
Is an analog to the compound of Example 6 prepared, and the final purification of the product is performed by column chromatography.

Claims (23)

A method for acylating an amino group of a peptide or protein to obtain an N-acylated peptide or protein comprising the following steps:
(A) A peptide or protein having at least one free amino group is represented by the following general formula (I):

{Where,
n is 0-8;
R ¹ is COOR ⁴ ;
R ² is a lipophilic moiety characterized by having a solubility in water of 20 ° C. in the range of 0.1 mg / 100 ml water to 250 mg / 100 ml water;
R ³ is an ester functionalized in the form of a carboxylic acid group, together with a carbonyl group to which R ³ is attached, and is a reactive ester or amino ester used for acylation of an amino group Represents a reactive N-hydroxy imide ester; and R ⁴ is hydrogen. }
And reacting in a mixture of an aprotic polar solvent and water under basic conditions, wherein the peptide or protein is Arg ²⁶ -GLP-1 (7-37) ^{; Arg 34 -GLP-1 (7-37} ); Lys 36 -GLP-1 (7-37); Arg 26,34 Lys 36 -GLP-1 (7-37); Arg 26,34 Lys 38 GLP-1 ^{(7-38); Arg 26,34 Lys 39} -GLP-1 (7-39); Arg 26,34 Lys 40 -GLP-1 (7-40); Arg 26 Lys 36 -GLP-1 (7-37 ^{); Arg 34 Lys 36 -GLP-} 1 (7-37); Arg 26 Lys 39 -GLP-1 (7-39); Arg 34 Lys 40 -GLP-1 (7-40); Arg ^{6,34 Lys 36,39 -GLP-1 (7-39} ); Arg 26,34 Lys 36,40 -GLP-1 (7-40); Gly 8 Arg26-GLP-1 (7-37); Gly 8 ^{Arg 34 -GLP-1 (7-37)} ; Gly 8 Lys 36 -GLP-1 (7-37); Gly 8 Arg 26,34 Lys 36 -GLP-1 (7-37); Gly 8 Arg 26,34 ^{Lys 39 -GLP-1 (7-39)} ; Gly 8 Arg 26,34 Lys 40 -GLP-1 (7-40); Gly 8 Arg 26 Lys 36 -GLP-1 (7-37); Gly 8 Arg 34 Lys ³⁶ -GLP-1 (7-37); Gly ⁸ Arg ²⁶ Lys ³⁹ -GLP-1 (7-39); Gly ⁸ Arg ³⁴ Lys ⁴⁰ -GLP-1 (7 ^{-40); Gly 8 Arg 26,34 Lys} 36,39 -GLP-1 (7-39); Gly 8 Arg 26,34 Lys 36,40 -GLP-1 (7-40); Arg 26,34 Lys 38 ^{GLP-1 (7-38); Arg} 26,34 Lys 39 GLP-1 (7-39); Arg 26,34 Lys 40 GLP-1 (7-40); Arg 26,34 Lys 41 GLP-1 (7 Arg ^26,34 Lys ⁴² GLP-1 (7-42); Arg ^26,34 Lys ⁴³ GLP-1 (7-43); Arg ^26,34 Lys ⁴⁴ GLP-1 (7-44); Arg ^26,34 Lys ⁴⁵ GLP-1 (7-45); Arg ^26,34 Lys ³⁸ GLP-1 (1-38); Arg ^26,34 Lys ³⁹ GLP-1 (1 Arg ^26,34 Lys ⁴⁰ GLP-1 (1-40); Arg ^26,34 Lys ⁴¹ GLP-1 (1-41); Arg ^26,34 Lys ⁴² GLP-1 (1-42); Arg ^26,34 Lys ⁴³ GLP-1 (1-43); Arg ^26,34 Lys ⁴⁴ GLP-1 (1-44); Arg ^26,34 Lys ⁴⁵ GLP-1 (1-45); Arg ^26,34 Lys ³⁸ GLP-1 (2-38); Arg ^26,34 Lys ³⁹ GLP-1 (2-39); Arg ^26,34 Lys ⁴⁰ GLP-1 (2-40); Arg ^26,34 Lys ⁴¹ GLP-1 (2 ^{-41); Arg 26,34 Lys 42 GLP} -1 (2-42); Arg 26,34 Lys 43 GLP-1 (2-43); Arg 26,34 Lys ^{4 GLP-1 (2-44);} Arg 26,34 Lys 45 GLP-1 (2-45); Arg 26,34 Lys 38 GLP-1 (3-38); Arg 26,34 Lys 39 GLP-1 ( ^{3-39); Arg 26,34 Lys 40 GLP} -1 (3-40); Arg 26,34 Lys 41 GLP-1 (3-41); Arg 26,34 Lys 42 GLP-1 (3-42); Arg ^26,34 Lys ⁴³ GLP-1 (3-43); Arg ^26,34 Lys ⁴⁴ GLP-1 (3-44); Arg ^26,34 Lys ⁴⁵ GLP-1 (3-45); Arg ^26,34 Lys ³⁸ GLP-1 (4-38); Arg ^26,34 Lys ³⁹ GLP-1 (4-39); Arg ^26,34 Lys ⁴⁰ GLP-1 (4-40); Arg ^26,34 Lys ⁴¹ GLP-1 (4-41); Arg ^26,34 Lys ⁴² GLP-1 (4-42); Arg ^26,34 Lys ⁴³ GLP-1 (4-43); Arg ^26,34 Lys ⁴⁴ GLP-1 (4-44); Arg ^26,34 Lys ⁴⁵ GLP-1 (4-45); Arg ^26,34 Lys ³⁸ GLP-1 (5-38); Arg ^26,34 Lys ³⁹ GLP-1 (5 Arg ^26,34 Lys ⁴⁰ GLP-1 (5-40); Arg ^26,34 Lys ⁴¹ GLP-1 (5-41); Arg ^26,34 Lys ⁴² GLP-1 (5-42); Arg ^{26,34 Lys 43 GLP-1 (5-43} ); Arg 26,34 Lys 44 GLP-1 (5-44); Arg 26,34 Lys 45 GLP-1 ( ^{-45); Arg 26,34 Lys 38 GLP} -1 (6-38); Arg 26,34 Lys 39 GLP-1 (6-39); Arg 26,34 Lys 40 GLP-1 (6-40); Arg ^{26,34 Lys 41 GLP-1 (6-41} ); Arg 26,34 Lys 42 GLP-1 (6-42); Arg 26,34 Lys 43 GLP-1 (6-43); Arg 26,34 Lys 44 GLP-1 (6-44); Arg ^26,34 Lys ⁴⁵ GLP-1 (6-45); Arg ²⁶ Lys ³⁸ GLP-1 (1-38); Arg ³⁴ Lys ³⁸ GLP-1 (1-38); ^{Arg 26,34 Lys 36,38 GLP-1 (} 1-38); Arg 26 Lys 38 GLP-1 (7-38); Arg 34 Lys 38 GLP-1 ( ^{-38); Arg 26,34 Lys 36,38 GLP} -1 (7-38); Arg 26,34 Lys 38 GLP-1 (7-38); Arg 26 Lys 39 GLP-1 (1-39); Arg ³⁴ Lys ³⁹ GLP-1 (1-39); Arg ^{26, 34} Lys ^{36, 39} GLP-1 (1-39); Arg ²⁶ Lys ³⁹ GLP-1 (7-39); Arg ³⁴ Lys ³⁹ GLP-1 ( ^{7-39); Arg 26,34 Lys 36,39 GLP} -1 (7-39); Arg 26 -GLP-1 (7-37), Arg 34 -GLP-1 (7-37), Lys 36 -GLP ^{-1 (7-37), Arg 26,34 Lys} 36 -GLP-1 (7-37), Arg 26 Lys 36 -GLP-1 (7-37), Arg 34 Lys 36 - GLP-1 (7-37), Gly ⁸ Arg ²⁶ -GLP-1 (7-37), Gly ⁸ Arg ³⁴ -GLP-1 (7-37), Gly ⁸ Lys ³⁶ -GLP-1 (7-37) ^{, Gly 8 Arg 26,34 Lys 36 -GLP} -1 (7-37), Gly 8 Arg 26 Lys 36 -GLP-1 (7-37); Gly 8 Arg 34 Lys 36 -GLP-1 (7-37) ^{; Arg 26 Lys 38 -GLP-1} (7-38), Arg 26,34 Lys 38 -GLP-1 (7-38), Arg 26,34 Lys 36,38 -GLP-1 (7-38), Gly ^{8 Arg 26 Lys 38 -GLP-1} (7-38); Gly 8 Arg 26,34 Lys 36,38 -GLP-1 (7-38); Gly 8, Arg 26,34, ^{Glu 37, Lys 38 -GLP-1} (7-38), Arg 26 Lys 39 -GLP-1 (7-39), Arg 26,34 Lys 36,39 -GLP-1 (7-39), Gly 8 Arg ^{26 Lys 39 -GLP-1 (7-39} ); Gly 8 Arg 26,34 Lys 36,39 -GLP-1 (7-39); Arg 34 Lys 40 -GLP-1 (7-40), Arg 26, ^{34 Lys 36,40 -GLP-1 (7-40} ), Gly 8 Arg 34 Lys 40 -GLP-1 (7-40), and ^{Gly 8 Arg 26,34 Lys 36,40 -GLP-} 1 (7-40 ). The R ³ represents an ester which, together with the carbonyl group to which R ³ is attached, represents a functional group suitable for acylation of a primary amine in the form of a carboxylic acid group. the method of. R ³ together with the carbonyl group where R ³ is attached to it, represents a reactive ester, A method according to claim 1 or 2.   The method of claim 3, wherein the reactive ester is a 1-hydroxybenzotriazole ester. R ³ together with the carbonyl group where R ³ is attached thereto, represent a reactive N- hydroxy imide ester method according to claim 1 or 2.   6. The method according to claim 5, wherein the imide part of the N-hydroxy imide ester is succinimide or phthalimide.   The process according to any of claims 1 to 6, wherein the mixture of aprotic solvent and water is a 1: 5 to 5: 1 mixture of N-methyl-2-pyrrolidone and water.   The aprotic polar solvent is selected from anhydrous tetrahydrofuran (THF), anhydrous dimethylformamide (DMF), acetone, dichloromethane, dimethyl sulfoxide (DMSO), dioxane, dimethylacetamide, and N-methyl-2-pyrrolidone, and mixtures thereof The method according to claim 1.   9. A process according to claim 8, wherein the aprotic polar solvent is selected from dimethylformamide, dimethyl sulfoxide, dimethylacetamide, and N-methyl-2-pyrrolidone.   The process according to claim 9, wherein the aprotic polar solvent is N-methyl-2-pyrrolidone.   The method according to any of claims 1 to 10, wherein the pH of the reaction mixture in step (a) is in the range of 9-13.   The method according to any of claims 1 to 10, wherein the pH of the reaction mixture in step (a) is in the range of 9.5 to 12.5.   The method according to any of claims 1 to 10, wherein the pH of the reaction mixture in step (a) is in the range of 10-12.   The method according to any one of claims 1 to 13, wherein the temperature of the reaction mixture in step (a) is in the range of 0 to 50C. R ² is characterized by having a solubility in 20 ° C. water in the range of 0.3 mg / water 100Ml～75mg / water 100 ml, A method according to any one of claims 1 to 14. R ² is _{C 3-39} - _{alkyl, C 3-39} - _{alkenyl, C 3-39} - alkadienyl, and are selected from steroidal residues A method according to any one of claims 1 to 15. R ² is heptyl, nonyl, undecanyl, tridecanyl, pentadecanyl, heptadecanyl, nonadecanyl, Ritokoroiru, deoxy roller yl, and selected from Koroiru The method of claim 16. R ² is heptyl, nonyl, undecanyl, tridecanyl, pentadecanyl, selected heptadecanyl, and nonadecanyl The method of claim 17. R ² is _{C 7-25} - is selected from alkyl, The method of claim 16. The following general formula (I):

{Where,
n is 0-8;
R ¹ is COOH;
R ² is a lipophilic moiety; and R ³ together with the carbonyl group to which R ³ is attached, 1-hydroxybenzotriazole ester , or reactive N-hydroxy imide・ Represents an ester}
A compound represented by   21. The compound of claim 20, wherein the 1-hydroxybenzotriazole ester is 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate. n is 0 or 1, R ³ together with the carbonyl group of where R ³ is attached thereto, represent a reactive N- hydroxy imide ester compound according to claim 20. R ² is _{C 3-39} - _{alkyl, C 3-39} - _{alkenyl, C 3-39} - alkadienyl and are selected from steroidal residues A compound according to any one of claims 20 to 22.