WO2006008238A1

WO2006008238A1 - Method for selective acylation

Info

Publication number: WO2006008238A1
Application number: PCT/EP2005/053207
Authority: WO
Inventors: Claus Ulrich Jessen; Louis Brammer Hansen
Original assignee: Novo Nordisk A/S
Priority date: 2004-07-16
Filing date: 2005-07-05
Publication date: 2006-01-26

Abstract

The present invention provides an effective and simple method for selectively acylating insulin or insulin analogues or precursors thereof at a free T-amino group in a lysine amino acid residue in the insulin molecule. The acylating reaction is conducted at mild reaction conditions at neutral pH and at a low water content in the acylation reaction mixture.

Description

METHOD FOR SELECTIVE ACYLATION

FIELD OF THE INVENTION

The present invention relates to a method for selective acylation of a free ε-amino group in insulin or insulin analogues and precursors thereof.

BACKGROUND OF THE INVENTION

Many diabetic patients are treated with multiple daily insulin injections in a regimen comprising one or two daily injections of a protracted insulin to cover the basal requirement supplemented by bolus injections of a rapid acting insulin to cover the requirement related to meals. Many diabetic patients are treated with multiple daily insulin injections in a regimen comprising one or two daily injections of a protracted insulin to cover the basal requirement supplemented by bolus injections of a rapid acting insulin to cover the requirement related to meals.

A class of compounds suitable for this task is insulin derivatives in which the ε- amino group in the lysine residue in position 29 of the B-chain is acylated with a hydrophobic moiety are disclosed in EP patents 792,290 and 894,095 and in US patent Nos. 5,693,609, 5,646,242, 5,922, 675, 5,750,497 and 6,011 ,007.

Human insulin and closely related insulins have three primary amino groups in the molecule namely the α-amino groups of Gly^A1 and Phe^B1, respectively, and the ε-amino group of Lys^B29. N-Acylation of unprotected insulin may - depending on the conditions - lead to a complex mixture of mono-, di- and even triacylated products. However, although a cer¬ tain preference for acylation of a specific position can often be observed the preference is not always sufficiently pronounced to make such direct acylation useful as a method of produc¬ ing monoacylated insulins since the formation of the desired product may be accompanied by the formation of considerable amounts of closely related by-products. When by-products are formed, this happens at the expense of the desired product and may lead to problems in the purification of the desired product.

Acylation of only one or two specific amino groups in the insulin molecule can be achieved if a suitably protected intermediate is available. A suitable intermediate can be an insulin derivative in which the amino group(s) not to be acylated is (are) blocked with a pro- tection group which can be removed selectively after the acylation has been performed. Such a protected intermediate can either be an insulin precursor or an insulin derivative in which it has been possible posttranslationally to introduce one or two protection groups in a specific way. For economic reasons, it is however very attractive to avoid the use of specific protec¬ tion groups if possible.

Selective acylation of in particular a free ε-amino group in either position B29 or in position B28 is disclosed on US patent No. 5,646,242 which discloses a method for selective acylation at pH above 9 by use of an activated ester of a fatty acid. US patent No. 5,905,140 discloses selective acylation of a free ε-amino group in insulin by used of an activated amide at basic pH.

The present invention is related to an improved process for obtaining high yields of insulin being acylated in an ε-amino group, in particular the ε-amino group in Lys^B29.

SUMMARY OF THE INVENTION

In its broadest aspect the present invention is related to a method for selectively acylation of a free ε-amino group of a lysine residue in insulin or insulin analogues or precur¬ sors thereof by reacting the insulin in question with an acylation agent under neutral condi¬ tions, i.e. without addition of a base, and at a water content in the reaction mixture of below about 5% w/w.

In one embodiment the water content in the reaction mixture is from about 0.01 to about 5% w/w.

In another embodiment the water content in the reaction mixture is from about 0.01 to about 4% w/w or from about 0.01 to about 2.5 w/w. In a further embodiment the water content in the reaction mixture is from about 0.1 to about 5, from about 0.1 to about 4,from about 0.1 to about 2.5% w/w or from about 0.1 to about 2% w/w .

In a still further embodiment the water content in the reaction mixture is from about 0.2 to about 5, from about 0.2 to about 4, from about 0.2 to about 2.5 or from about 0.2 to about 2% w/w.

In one aspect of the present invention the acylation agent is an activated ester or an activated amide. In another aspect the activated ester is an ONSu ester and in a still further aspect the activated ester is an ONSu ester of a fatty acid.

In one aspect of the present invention the activated amide is an azolide of the acid corresponding to the acyl group to be introduced and in a further aspect the azolide is benzotri- azole or substituted benzotriazole.

The acyl group may be selected from the group consisting of unbranched, aliphatic monocarboxylic acids having from 6 to 24 carbon atoms, dicarboxylic acids and lithocholic ac¬ ids and derivatives thereof. In one embodiment of the present invention the acyl group is a fatty acid and in par¬ ticular a long chain fatty acid having from 6 to 18 C-atoms, from 10 to 18 C-atoms or from 10 to 14 C-atoms.

In still another embodiment the fatty acid is selected from the group consisting of capric acid, lauric acid, tetradecanoic acid (myristic acid), pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, dodecanoic acid, tridecanoic acid, and tetradecanoic acid.

In one embodiment the acylated insulin is N^εB29tetradecanoyl-des(B30) human insu¬ lin.

In another embodiment the acylated insulin is N^εB29-lithocholoyl-γ-glutamyl des(B30) human insulin.

In still another embodiment the acylated insulin is N^εB29-(N^α-(HOOC(CH₂)₁₄CO)-γ- GIu) des(B30) human insulin or N^εB29-(N^α-(HOOC(CH₂)i₆CO)-γ-Glu) des(B30) human insulin.

The method according to the present invention may comprise the following steps: a) dissolving insulin or an insulin analogue or a precursor thereof in a polar organic solvent; b) adding an activated acylation agent and conducting the acylation at neutral pH at a tem¬ perature from about -2O⁰C to about 5O⁰C and at a water content in the reaction mixture below about 5% w/w at neutral pH; and c) isolating the product being acylated in the desired position.

The temperature may vary within a broad range. Suitable temperature ranges for step b) are ranges from about -20⁰C to about 5O⁰C, from about -20⁰C to about 40⁰C, from about -20°_tC to about 30⁰C, from about 0⁰C to about 50⁰C, from about 0⁰C to about 40⁰C, from about 0⁰C to about 35⁰C, from about 2O⁰C to about 50⁰C, from about 2O⁰C to about 4O⁰C and from about 20⁰C to about 35⁰C.

The starting insulin product to be acylated may typically be dried in a well known manner before it is acylated in the method according to the invention. Thus in one embodi¬ ment of the present invention the insulin or insulin analogue or a precursor thereof is dried to a water content of less that about 10% w/w before being dissolved in the reaction medium (step a) and in another embodiment the insulin or insulin analogue or a precursor thereof is dried to a water content of about 4 to about 5% w/w before being dissolved in the reaction mixture (step a).

Thus the method according to the present invention may comprise the following steps

1) drying the starting insulin product to a water content of less that about 10% w/w;

2) dissolving the dried compound in a polar organic solvent; 3) acylating the insulin starting product at neutral pH with an activated acylating agent at a temperature from about -2O⁰C to about 50°C and at a water content in the reaction mixture of between about 0.01 and about 5% w/w and

4) isolating the insulin or insulin analogue or precursor thereof acylated in the de- sired position.

As an alternative of drying the starting insulin product a suitable drying agent may be added to the reaction mixture before adding the acylation agent. Thus in another em¬ bodiment of the present invention a chemical desiccant is added before the acylation step (step b) to reduce the water content to below about 5 or about 2.5% w/w. In still another embodiment the solution from step a) is passed through a physical desiccant before step b) to reduce the water content to below about 5 to 2.5% w/w.

In one embodiment the physical desiccant can be a molecular sieve or a silica gel.

The polar organic solvent may be any suitable solvent such as DMSO, DMF, NMP and sulpholane.

DEFINITIONS

The term "insulin" is intended to include human insulin, porcine insulin, insulin ana¬ logues and insulin derivatives derived from insulin and having the molecular structure similar to that of human insulin including the disulfide bridges between Cys^A7 and Cys^B7 and be¬ tween Cys^⁰ and Cys^B19 and an internal disulfide bridge between Cys^A6 and Cys^A11 to pre- serve the insulin activity.

An insulin analogue is an insulin molecule having one or more mutations, substitu¬ tions, deletions and or additions of the A and/or B amino acid chains relative to the human insulin molecule. The insulin analogues are preferably such wherein one or more of the natu¬ rally occurring amino acid residues, preferably one, two, or three of them, have been substi- tuted by another codable amino acid residue. Thus position 28 of the B chain may be modi¬ fied from the natural Pro residue to one of Asp, Lys, or He. In another embodiment Lys at po¬ sition B29 is modified to Pro. Also, Asn at position A21 may be modified to Ala, GIn, GIu, GIy, His, He, Leu, Met, Ser, Thr, Trp, Tyr or VaI, in particular to GIy, Ala, Ser, or Thr and pref¬ erably to GIy. Furthermore, Asn at position B3 may be modified to Lys. Further examples of insulin analogues are insulin analogues wherein PheB1 or ThrB30 have been deleted; insulin analogues wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogues wherein the A-chain and/or the B-chain have a C-terminal extension. Thus one or two Arg may be added to position B1. By "insulin precursor" is meant a single-chain polypeptide which by one or more subsequent chemical and/or enzymatic processes can be converted into insulin. An example of a precursor is an insulin precursor with an amino acid sequence B(1 -29)-Ala-Ala-Lys-A(1 - 21) wherein A(1-21) is the A chain of human insulin and B(1-29) is the B chain of human in- sulin in which Thr(B30) is missing. This insulin precursor may be converted in human insulin by enzymatic cleaving off the Ala-Ala-Lys bridge which connects the amino acid residue in position B29 with the amino acid in position A21 , and enzymatic coupling of a Thr amino acid to the B29 amino acid residue. Other insulin precursors may comprise an N-terminal exten¬ sion to the B-chain which is then later on cleaved off by suitable enzymatic or chemical treat- ment, see US patent No. 6521738, WO 97/22706, WO 97/00581 and WO 00/04172.

By "connecting peptide" or "C-peptide" is meant the connection moiety "C" of the B-

C-A polypeptide sequence of a single chain preproinsulin-like molecule. Specifically, in the natural insulin chain, the C-peptide connects position 30 of the B chain and position 1 of the

A chain. A "mini C-peptide" or "connecting peptide" such as those described herein, connect B29 or B30 to A1 , and differ in sequence and length from that of the native human C-peptide.

With "desB30" or "B(1-29)" is meant a natural insulin B chain lacking the B30 amino acid residue, "A(1-21)" means the natural insulin A chain, "B(1-27)" means the natural B chain lacking the B28, B29, and B30 amino acid residues etc.

With "preferential" or "selective acylation" is meant an acylation which occurs in a de- sired position at a higher degree, preferably at least at two or three times higher degree than in a not desired position. In the method according to the present invention acylation should preferably only take place in the ε-amino group in the Lys^B29 and not in the two N-terminal α- amino groups at the two chain insulin precursor. The ratio between the mono acylated

LysB29 and mono acylated A1 and B1 should be higher than about 4 to about 1, preferably higher than about 10 to about 1.

The term "activated" acylation agent means an acylation agent which has been acti¬ vated using general techniques described in Methods of Enzymology 25, 494-499 (1972). "Neutral" pH is meant to cover a pH value of from about 6.5 to about 8.

DESCRIPTION OFTHE INVENTION The present method provides a method of securing a selective acylation in a desired position of an insulin molecule. By conducting the acylation reaction in a reaction mixture with a low water content it has surprisingly been shown that a selective acylation can be ob¬ tained at neutral pH. Neutral pH is advantageous because the less harsh conditions have turned out to give an impurity profile of the acylation reaction mixture which enables a much easier purification and isolation of the desired selectively acylated product from non reacted acylating agent, insulin mono acylated in B1 or A1 and di- and triacylated insulin by-products. Thus, simple chromatography steps enable effective separation of the selectively acylated product from undesired by-products. To ensure a high selectivity the water content in the acylation reaction has to be kept at a low content and good results are achieved when the water content in the acylation reaction mixture is between about 0.1 and about 5% w/w.

An adequately low water content in the reaction mixture con be obtained by either using reactants with a low water content or to remove the excessive water from one or more of the reactants before the acylation reaction.

To ensure such low water content in the reaction mixture the insulin starting product to be acylated can be dried to a suitable water content. Drying of the insulin starting product can be conducted in any convenient way but is typically conducted in vacuum at room tem¬ perature. Alternatively the insulin starting product can be dried under conventional freeze drying conditions.

The insulin starting product is typically dried to a water content of less than about 10 % w/w, in particular to a water content below about 6-8% w/w and preferably to a water con¬ tent as low as about 4-5% w/w before it is acylated by the claimed method that is before it is dissolved in the polar solvent in step a). The low water content in the reaction mixture can also be obtained by use of a suit¬ able chemical desiccant, such as silyl compounds like trimethylchlorsilan, or by use of physi¬ cal desiccants such as molecular sieves or silica gels.

The polar organic solvent may be any suitable polar solvent such as DMSO, DMF, NMP and sulpholane and mixtures thereof. As even so-called anhydrous solvent as the polar organic solvents used in the present method still may contain a low amount of water and because it may be difficult to conduct the acylation reaction under conditions where only minimal water is absorbed from the surrounding it may also be advisable to add a desiccant during the acylation step.

In one embodiment of the invention, the acyl group to be introduced into the ε-amino group of a Lys residue is the acyl group of a monocarboxylic acid of the general formula (I):

M-COOH (I) wherein M is a long chain hydrocarbon group which may optionally be interrupted by one or more groups each independently selected from the group consisting of an oxygen atom and a sulphur atom. Specific examples of this type are the acyl group of an unbranched, aliphatic monocarboxyϋc acid having from 6 to 24 carbon atoms, in particular an acyl group selected from the group comprising CH₃(CH₂)_SCO-, CH₃(CH₂)₉CO-, CH₃(CHa)₁₀CO-, CH₃(CH₂)iiCO-, CH₃(CH₂)₁₂CO-, CH₃(CH₂)₁₃CO-, CH₃(CH₂)_I4CO-, C H₃(CH₂) ₁₅C0-, CH₃(CH₂)₁₆CO-, CH₃(CH₂)I₇CO-, CH₃(CH₂)₁₈CO-, CH₃(CH₂)₁₉CO-, CH₃(CH₂)₂₀CO-, CH₃(CH₂)₂iCO- and CH₃(CH₂)₂₂CO-.

The acyl group to be introduced into the ε-amino group of a Lys may also be an acyl group of a dicarboxylic acid of the general formula (II):

HOOC-D-COOH (II)

wherein D is a long chain hydrocarbon group which may optionally be interrupted by one or more groups each independently selected from the group consisting of an oxygen atom and a sulphur atom. More preferred, the acyl group is one of the acyl groups of a dicarboxylic acid of the general formula (II) wherein D is an unbranched, divalent aliphatic hydrocarbon group having from 6 to 22 carbon atoms, in particular an acyl group selected from the group comprising HOOC(CH₂)₆CO-, HOOC(CH₂)₈CO-, HOOC(CH₂)₁₀CO-, HOOC(CH₂)₁₂CO-, HOOC(CH₂)_I4CO-, HOOC(CH₂)₁₆CO-, HOOC(CH₂)₁₈CO-, HOOC(CH₂)₂oCO- and HOOC(CH₂)₂₂CO-.

In further embodiment of the present invention, the acyl group to be introduced into the ε-amino group of a Lys residue is a group of the general formula (III):

CH₃(CH₂)_XCONHCH(COOR¹)CH₂CH₂CO- (III)

wherein x is an integer from 8 to 24 and R¹ is hydrogen or a group which can be exchanged with hydrogen after the acylation has been performed, for example methyl, ethyl or terf-butyl.

Preferred values of x are 10, 12 and 14. When the acylation has been performed and R¹ is different from hydrogen, the ester group of which R¹ is a part can be hydrolysed to give the corresponding free acid and the alcohol R¹OH by methods known per se. Thus, when R¹ is methyl or ethyl, the hydrolysis can be performed under alkaline conditions and when R¹ is tert- butyl, the hydrolysis can be carried out using trifluoroacetic acid.

In yet another embodiment the acyl group is connected to the lysine residue using an amino acid linker. According to this embodiment the acyl group is connected to a lysine residue via a γ- or an α-glutamyl linker, or via a β- or an α-aspartyl linker, or via an α-amido- γ-glutamyl linker, or via an α-amido-β-aspartyl linker. In a still further embodiment the acyl group is lithocholoyl-Glu(OH)-OR, wherein OH represent the free carboxyl group of the ^-carbon and OR is an ester group protecting the α-carbon of glutamic acid wherein R is methyl, ethyl, ortert. butyl.

In further embodiment of the invention, the acyl group to be introduced into the ε-amino group of a Lys residue is a group of the general formula (IV):

Iithocholoyl-NHCH(COOR²)CH₂CH₂CO- (IV)

wherein R² is hydrogen or a group which can be exchanged with hydrogen after the acylation has been performed, for example methyl, ethyl or fert-butyl. When the acylation has been performed and R² is different from hydrogen, the ester group of which R² is a part can be hydrolysed to give the corresponding free acid and the alcohol R²OH by methods known perse. Thus, when R² is methyl or ethyl, the hydrolysis can be performed under alkaline conditions and when R² is ferf-butyl, the hydrolysis can be carried out using trifluoroacetic acid. The acylating agent can be any suitable acylating agent capable of introducing an acyl group in the desired position in the insulin molecule. Suitable acylating agents are acti¬ vated esters and activated amides. Further suitable acylating agents are anhydrides and hal- ides.

Activated esters are well known and can be prepared using general techniques de- scribed in Methods of Enzymology 25, 494-499 (1972). The most commonly used activated ester type is ONSu esters.

The acylation according to the present invention may also be carried out using an acylating agent which is an activated amide, more particularly an azolide of the acid corresponding to the acyl group to be introduced. Such azolides (1-acylazoles) can be prepared according to known methods, see for example Staab HA Angew. Chem. 74 (1962) 407-423. Examples of azoles which can be used in the preparation of the acylating agents of this invention are pyrazole, imidazole, 1 ,2,3-triazole, 1 ,2,4-triazole, tetrazole, phenyltetrazole and - where possible - the corresponding benzanelated compounds e.g. 1 H-indazol, benzimida∑ole and benzotriazole. Preferred azoles for the preparation of the acylating agent are 1 ,2,4-triazole, benzotriazole and substituted benzotriazoles. Optionally, the azoles mentioned can be substituted with one or more substituents selected from the group comprising alkyl (Ci - C₅, branched or unbranched, in particular methyl, ethyl, propyl and isopropyl), halogen (e.g. fluoro, chloro and bromo, in particular fluoro and chloro), nitro, alkoxy (Ci - C₅, branched or unbranched, in particular methoxy, ethoxy, propoxy and isopropoxy), dialkylated amino (C₁ - C₅, branched or unbranched), sulphonic acid, carboxy and alkoxycarbonyl (Ci - C₅, branched or unbranched). A preferred group of acylating agents are 1 -acyl benzotriazoles and a preferred acylating agent is 1 -tetradecanoyl benzotriazole. Other preferred 1 -acyl benzotriazoles are such which are derived from mono- or disubstituted benzotriazoles, e.g. 5-substituted or 6-substituted or 5,6-disubstituted benzotriazoles such as 5-methylbenzotriazole, 5-chloroben∑otriazole, 6- chloroben∑otria∑ole, 5-nitroben∑otriazole, 5,6-dimethylbenzotriazole and 5,6-dichlorobenzo- triazole.

Basically, the lower limit of the temperature at which the acylation can be carried out is determined by the freezing point of the medium while the upper limit is determined by the temperature at which the starting insulin or the acylated insulin will deteriorate. This again will depend Ia. on the composition of the medium. Thus, while it may be possible to carry out the reaction at temperatures between about -20 ⁰C and about 50 ⁰C it is usually most convenient to carry out the reaction at a temperature between about -5 ⁰C and about 3O⁰C.

The starting product for the acylation, the insulin or insulin analogue or a precursor thereof can be produced by either well-know peptide synthesis or by well known recombinant production in suitable transformed microorganisms.

A human insulin precursor B(1 -29)-Ala-Ala-Lys-A(1 -21 ) may e.g. be produced in yeast as disclosed in US patent No. 4,916,212. This insulin precursor can then be converted into desB30 human insulin by ALP cleavage of the Ala-Ala-Lys peptide chain to give desB30 human insulin which can then be acylated according to the present method. Isolation and purification of the acylated products prepared by the method of the present invention can be carried out by methods known per se, including gel filtration, crystallization and chromatography.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each refer- ence were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the inven¬ tion. The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subject matter re- cited in the claims appended hereto as permitted by applicable law.

EXAMPLES

Example 1

Drying of desB30 human insulin. Sodium acetate precipitated des(B30) human insulin (58.6 g) was dried for 3 days at

22-24⁰C under vacuum (5-10 hP). Yield: 19.4 g (33%). Water content determined by Karl Fischer to 4.7% w/w.

Example 2 Synthesis of N^εB29-tetradecanoyl des(B30) human insulin.

Des(B30) human insulin (4.0 g,~0.60 mmol) with a water content of 4.7% w/w was dissolved in dimethylsulfoxide (170 ml) at 22⁰C. The pH of the solution was checked using water wet indicator paper and found to be between 6 and 7. Tetradecanoic acid 2,5-dioxo- pyrrolidin-1-yl ester (ONSu ester) (0.22g (0.066mmol) dissolved in 12 ml dimethylsulfoxide) was added within 3 minutes. The reaction mixture was stirred at 22⁰C for 30 minutes and then quenched by adding water (170 ml). The reaction mixture was analyzed by RP-HPLC with the following results:

Starting material: 11% N^αA1-tetradecanoyl des(B30) human insulin 2%

N^εB29-tetradecanoyl des(B30) human insulin: 69%

Di and tri acylated des(B30) human insulin 17 %

Example 3 Synthesis of N^εB29-N-lithocholyl-glutamo-5-yl methyl ester des(B30) human insulin.

Des(B30) human insulin (1.16 g, -0.18 mmol) with a water content of 4,7% w/w was dissolved in dimethylsulfoxide (60 ml) at 22⁰C. The pH of the solution was checked using wa¬ ter wet indicator paper and found to be between 6 and 7. N-lithocholyl-glutamo-5-yl methyl ester O-hydroxy succinimide (0.119g (0.019mmol) was added in one portion. The reaction mixture was stirred at 22⁰C for 10 minutes. An in-process sample showed the following re¬ sults:

Starting material: 8% N^αA1- lithocholoyl-γ-glutamyl des(B30) human insulin 2%

N^εBZ9- lithocholoyl-γ-glutamyl des(B30) human insulin: 73%

Di and tri acylated des(B30) human insulin 17 %

Example 4 Synthesis of N^εB29-tetradecanoyl des(B30) human insulin.

Des(B30) human insulin (1.2 g,~0.20 mmol) with a water content of about 5% w/w was dissolved in dimethylsu If oxide (21 ml) at about 2O⁰C. The pH of the solution was checked using water wet indicator paper and found to be between 6 and 7. Tetradecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (74 mg ~0.23mmol) was added. The reaction mixture was stirred at 22⁰C for 1 hour and then quenched by adding water. The reaction mixture was ana¬ lyzed by RP-HPLC with the following results:

Starting material: 5%

N^αA1-tetradecanoyl des(B30) human insulin 3% N^Eβ29-tetradecanoyl des(B30) human insulin: 59%

Di and tri acylated des(B30) human insulin 33 %

Example 5

Synthesis of N^εB29-tetradecanoyl des(B30) human insulin. Des(B30) human insulin (1.2 g,~u-20 mmol) with a water content of about 5% w/w was dissolved in dimethylsu If oxide (21 ml) at about 2O⁰C. The pH of the solution was checked using water wet indicator paper and found to be between 6 and 7. 5-chloro-1- tetradecanoylbenzotriazole (83 mg ~0.23mmol) was added. The reaction mixture was stirred at 22⁰C for 1 hour and then quenched by adding water. The reaction mixture was analyzed by RP-HPLC with the following results:

Starting material: 8%

N^αA1-tetradecanoyl des(B30) human insulin 3%

N^εB29-tetradecanoyl des(B30) human insulin: 56% Di and tri acylated des(B30) human insulin 27 % Example 6

Des(B30) human insulin was dried for 6 days at 22-24⁰C under vacuum (5-10 hP). The water content determined by Karl Fischer was found to be 1.4% w/w.

Example 7

Synthesis of N^εB29-tetradecanoyl des(B30) human insulin.

Des(B30) human insulin (1.0 g, ~0.15 mmol) with a water content of 1.4% w/w was dissolved in dimethylsulfoxide (43 ml) at 21 ⁰C. The pH of the solution was checked using wa- ter wet indicator paper, and found to be between 7.5 and 8. An aliquot of the solution was diluted with tree times the volume of pH 7.0 demineralised water. The pH of the solution was found to be 7.91 using a calibrated pH-electrode. Acetic acid (81 microliter of a 40.6 milli mo¬ lar acetic acid in dimethylsulfoxide, ~0.15 mmol) was added, and the pH of the solution was found to be 7.20 using a calibrated pH-electrode. Tetradecanoic acid 2,5-dioxo-pyrrolidin-1 -yl ester (64 mg, ~0.20mmol dissolved in 3 ml dimethylsulfoxide) was added within 3 minutes. The reaction mixture was stirred at RT for 45 minutes, and then quenched by adding water (43 ml).

The reaction mixture was analyzed by RP-HPLC with the following results:

Starting material: „, 5.6%

N^A1-tetradecanoyl des(B30) human insulin 1.6%

N^εBZ9-tetradecanoyl des(B30) human insulin: 60%

Di and tri acylated des(B30) human insulin 32 %

Example 8

Synthesis of N^εB29-tetradecanoyl des(B30) human insulin.

Des(B30) human insulin (1.0 g, ~0.15 mmol) with a water content of 1.4% w/w was dissolved in dimethylsulfoxide (43 ml) at 22°C. Acetic acid (240 microliter of a 40.6 milli molar acetic acid in dimethylsulfoxide, -0.45 mmol) was added. An aliquot of the slightly cloudy so¬ lution was diluted with three times its volume of water, and the pH was found to be 6.0-6.5 using wet indicator paper, and 6.48 using a calibrated pH-electrode. Tetradecanoic acid 2,5- dioxo-pyrrolidin-1 -yl ester (64 mg, ~0.20mmol dissolved in 3 ml dimethylsulfoxide) was added within 3 minutes. The reaction mixture was stirred at RT for 45 minutes, and then quenched by adding water (43 ml). The reaction mixture was analyzed by RP-HPLC with the following results:

Starting material: 4.6%

N^A1-tetradecanoyl des(B30) human insulin 2.2%

N^εB29-tetradecanoyl des(B30) human insulin: 57%

Di and tri acylated des(B30) human insulin 35 %

Example 9

Synthesis of N^εBZ9-tetradecanoyl des(B30) human insulin.

Des(B30) human insulin (1.0 g, ~0.15 mmol) with a water content of 1.4% w/w was dissolved in dimethylsulfoxide (43 ml) at 33-36⁰C. Tetradecanoic acid 2,5-dioxo-pyrrolidin-1 - yl ester (54 mg, ~0.17mmol dissolved in 3 ml dimethylsulfoxide) was added within 3 minutes. The reaction mixture was stirred at 33-36°C for 45 minutes, and then quenched by adding water (43 ml).

The reaction mixture was analyzed by RP-HPLC with the following results: Starting material: 7.5%

N^A1-tetradecanoyl des(B30) human insulin 1.4% N^εB29-tetradecanoyl des(B30) human insulin: 75%

Di and tri acylated ,des(B30) human insulin 11 %

Example 10

Synthesis of N^εB29-tetradecanoyl des(B30) human insulin. Des(B30) human insulin (1.0 g, ~0.15 mmol) with a water content of 1.4% was dis¬ solved in dimethylsulfoxide (43 ml) and demineralised water (2.0 ml) at 25⁰C. Tetradecanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (64 mg, ~0.20mmol dissolved in 3 ml dimethylsulfoxide) was added within 3 minutes. The reaction mixture was stirred at RT for 45 minutes, and then quenched by adding water (43 ml). The reaction mixture was analyzed by RP-HPLC with the following results:

Starting material: 7.3%

N^A1-tetradecanoyl des(B30) human insulin 3.2%

N^εB29-tetradecanoyl des(B30) human insulin: 61 % Di and tri acylated des(B30) human insulin 28 % The analytical reverse phase HPLC (RP-HPLC) system consisted of a LiChroSpher column run at 50⁰C using an acetonitrile/water gradient at pH2.5 and recorded at 214 nm. The obtained conversions are expressed as area percentage.

Example 11

Purification of acylated Des-B30 insulins

Purification of the acylated Des-B30 insulins is carried out using anion exchange chromatography (1 x25 cm column) with Source 3OQ (polystyrene based mono disperses 30 μm particle) from Amersham Bioscience. The acylation mixture is diluted with one volume of water and applied on the anion exchange column. The column is pre-equilibrated in an etha- nol containing Tris buffer at neutral pH. Unbound material is washed out of the column and bound components are subsequently eluted using a linear gradient of ammonium acetate in ethanol and Tris. Fractions are collected and analysed for HPLC-purity. Pure fractions are pooled and analysed for purity and concentration to determine product purity and step yield.

HPLC analysis

The method is based on a RP-HPLC method at pH 2.5 using a mobile phase com- prised of ammonium sulphate. Separation of components is as carried out using an isocratic elutioji with acetonitrile as organic modifier. Detection of components is ,done at 214 nm. Pu¬ rity of acylated Des-B30 and the content of impurities are given in percentage by area. De¬ termination of the compound concentration is done using an internal standard.

Claims

1. A method of selectively acylating insulin or an insulin analogue or a precursor thereof having a free ε-amino group in a Lys residue contained therein, which method comprises reacting the ε- amino group with an acylating agent at neutral pH and at a water content in the reaction mixture of below about 5% w/w.

2. Method according to claim 1 , wherein the water content in the reaction mixture is from about 0.01 to about 5% w/w.

3. Method according to claim 1 , wherein the water content in the reaction mixture is from about 0.01 to about 4% w/w.

4. Method according to claim 1 , wherein the water content in the reaction mixture is from about 0.1 to about 4% w/w.

5. Method according to claim 1 , wherein the water content in the reaction mixture is from about 0.1 to about 2.5% w/w.

6. Method according to claim 1-5, wherein the acylation agent is an activated ester or an ac¬ tivated amide.

7. Method according to claim 6, wherein the activated ester is an ONSu ester.

8. Method according to claim 6, wherein the activated amide is an azolide of the acid corre¬ sponding to the acyl group to be introduced.

9, Method according to claim 8, wherein the azolide is benzotriazole or substituted benzotria- zole.

10. Method according to claim 1 , wherein the acyl group is selected from the group consisting of an acyl group of an unbranched, aliphatic monocarboxylic acid having from 6 to 24 carbon atoms, an acyl group of a dicarboxylic acid and a lithocholic acid group or a derivative thereof.

11. Method according to claim 10, wherein the acyl group is a fatty acid acyl group.

12. Method according to claim 11 , wherein the fatty acid has from 6 to 18 C-atoms.

13. Method according to claim 11 , wherein the fatty acid has from 10 to 18 C-atoms or from 10 to 14 C-atoms.

14. Method according to claim 1 , wherein the acylated insulin is N^εB29tetradecanoyl-des(B30) human insulin.

15. Method according to claim 1 , wherein the acylated insulin is N^εB29-lithocholoyl-γ-glutamyl des(B30) human insulin.

16. Method according to claim 1 , wherein the acylated insulin is N^εB29-(N^α- (HOOC(CH₂)i₄CO)-γ-Glu) des(B30) human insulin or N^εB29-(N^α-(HOOC(CH₂)₁₆CO)-γ-Glu) des(B30) human insulin.

17. Method according to any of the preceding claims 1-16 comprising the following steps: a) dissolving insulin or an insulin analogue or a precursor thereof in a polar organic solvent; b) adding an activated acylation agent and conducting the acylation at neutral pH at a tem- perature from about -2O⁰C to about 5O⁰C and at a water content in the reaction mixture below about-5% w/w at neutral pH; and c) isolating the product being acylated in the desired position.

18. Method according to claim 17, wherein the acylation is conducted at a temperature oτ from about -20⁰C to about 40⁰C.

19. Method according to any of claim 1-18, wherein the polar organic solvent is selected from the group consisting of dimethylsu If oxide, DMF, NMP and sulpholane and mixtures thereof.

20. Method according to any of claims 1-19, wherein the insulin or insulin analogue or a pre¬ cursor thereof is dried to a water content of less that about 10% w/w before being the acyla¬ tion reaction.

21. Method according to claim 20, wherein the insulin or insulin analogue or precursors thereof is dried to a water content of about 4 to about 5% w/w before the acylation reaction.

22. Method according to claim 17, wherein a chemical desiccant is added before step b) or the solution from step a) is passed through a physical desiccant before step b) to reduce the water content to below about 5% w/w or to below about 2.5% w/w.

23. Method according to claim 22, wherein the physical desiccant is a molecular sieve or a silica gel.

24. Method according to claim 17, wherein step c) comprises at least one chromatography step.

25. Method according to claim 17, wherein step c) comprises an HPLC or an ion exchange step or a combination thereof.