WO1995026975A9

WO1995026975A9 - Acyclic aliphatic branched alkyl groups as constituents of protective groups bound to side chains of amino-acid moieties, and their use in the preparation of peptide, polypeptide or protein structures

Info

Publication number: WO1995026975A9
Application number: PCT/SE1995/000337
Authority: WO
Filing date: 1995-03-30
Publication date: 1995-12-07

Abstract

A protective group bound to a side chain of an amino-acid moiety, which group is an aliphatic acyclic branched alkyl group protecting a thiol, hydroxyl, or carboxyl group or an aliphatic acyclic branched alkoxycarbonyl group protecting an amino group or heterocyclic nitrogen, wherein the alkyl and alkoxy entities have from 6 to 18 carbon atoms and have at least two identical alkyl chains, is described. Further, the use of such a protective group bound to a side chain of an amino-acid moiety in the synthesis of peptides, polypeptides and proteins is disclosed.

Description

Acyclic aliphatic branched alkyl groups as constituents of protective groups bound to side chains of amino-acid moieties, and their use in the preparation of peptide, polypeptide or protein structures.

The present invention relates to protective groups bound to side chains of amino-acid moieties, and their use in the preparation of peptide, polypeptide or protein structures. The protective groups of the invention comprise acyclic aliphatic branched alkyl groups.

Background of the invention

It is well known in the art that the preparation of peptides and proteins by chemical peptide synthesis is hampered by a wide range of side reactions which result in low yield and low purity of the product. These problems can be divided into problems associated with individual amino-acid residues employed in the synthesis, and problems associated with incomplete acylation of the N-terminal amino groups of the peptide chain. The first group of problems may result from the fact that the protective groups of the side chains employed in the synthesis are not sufficiently stable under the reaction conditions used during the synthesis, that the protective groups do not give sufficient protection against a side reaction, or that the removal of the protective groups give rise to specific side reactions.

The second group of problems is usually referred to as "sequence dependent" and is suggested to arise from the association of the peptide chain into non- covalently linked aggregates. In the synthesis of a polypeptide or protein in homogenous solution these problems will be reflected in low solubility of the polypeptide, whereas in solid-phase peptide synthesis aggregation will result in lower reaction rates in the coupling of protected amino acid or lower reaction rates in the deprotection of the temporary N protective group.

Description of prior art

Chemical peptide synthesis is commonly carried out with either of two strategies, namely using the acid labile tert- butyloxycarbonyl (Boc) or the base labile 9-fluorenylmethoxycarbonyl (Fmoc) group, respectively. These different strategies will require different protective groups on the side chains of the amino acids. These two strategies will in the following text be referred to as

Boc chemistry and Fmoc chemistry, respectively.

Many protective groups have been suggested to be used as side-chain protective groups in peptide synthesis. In the synthesis of peptides with amino acids protected at the N^α nitrogen with the Boc group, the most commonly used side-chain protecting groups are benzyl groups that are used as benzyl ester, benzyloxycarbonyl groups or benzyl ethers, or for some residues, other aromatics. In the synthesis of peptides with amino acids protected at the N^α nitrogen with Fmoc chemistry, the most commonly used protective groups are the fert- butyl ester, tert-butyl ether and fert- butyl carbamates.

Several of the side-chain protected amino-acid derivatives employed in peptide synthesis with Boc and Fmoc chemistry have protective groups that are insufficiently stable under the reaction conditions employed in standard synthesis protocols, or are insufficiently stable under modified reaction conditions. An important example of modified reaction conditions is the use of base-labile protective groups in the combined synthesis of polypeptides and proteins with Boc amino acids.

The β-COOH group of aspartic acid is during the synthesis according to Boc chemistry commonly protected with benzyl or cyclohexyl esters. Tarn et al reported in Tetrahedron Letters, 1979, No 42, pages 4033-4036, that the cyclohexyl protecting group was superior to the benzyl ester as protection against base-catalyzed aspartimide formation, and that a 24 hour treatment with triethylamine still gave rise to 14% aspartimide when protecting the β- COOH as a cyclohexylester. Similar results were obtained by Nicolas et al in Tetrahedron Letters, 1989, vol. 30, pages 497-500.

Yajima et al showed in Chem. Pharm. Bull., 1986, vol. 34 (10), pages 4356- 4361 that the β-A menthyl derivative of aspartic acid, when used in peptide synthesis, was more resistant against base-catalyzed aspartimide formation than the β-benzyl ester derivative. Treatment with triethylamine for 40 hours gave 3.9 % aspartimide for the β-/-menthyl ester and 36.7 % for the β-benzyl ester.

In a later study 1989 by Iguchi et al in Chem. Pharm. Bull. 37(8), pages 2209- 2211, the β-adamantyl derivative of aspartic acid was employed in peptide synthesis but no assessment of the protection against base-catalyzed aspartimide formation was given.

The tert-butyl esters used in Fmoc chemistry for the protection of the β-COOH group of aspartic acid has previously been thought not to give rise to aspartimide during treatment with piperidine. Recent reports 1994 by Dolling et al in J.Chem.Soc.Chem.Commun., pages 853-854) showed that, unexpectedly, high levels of aspartimide and aspartimide-related side products such as the piperidide derivative, was formed when synthesizing peptides having certain sequences with Fmoc chemistry using the tert-butyl ester as a protective group for aspartic acid.

It is clear from these reports that base-catalyzed aspartimide formation is a problem in the synthesis of polypeptides and proteins, and that new protective groups that provides a mean to prevent, or greatly reduce, the formation of such side products are welcomed in the field of peptide synthesis.

The side chain of histidine contains two heterocyclic nitrogens, one of which is usually protected during solid-phase peptide synthesis. It is well known in the art that the π-nitrogen in the imidazole nucleus can give rise to racemization by abstracting the C hydrogen when the COOH group is activated. In order to avoid this reaction, either electron-withdrawing groups has to be introduced at the τ-nitrogen, or a protective group has to be introduced at the π-nitrogen. In the latter case the protective group does not have to be electron-withdrawing. Colombo et al discussed in J.Chem. Soc. Perk. Trans. 1 , 1985, pages 1811- 1815, the problems associated with the introduction of protective groups on the imidazole nucleus of histidine, and it was concluded that the reaction between equimolar amounts of the nucleophilic imidazole nucleus of histidine and an electrophilic reagent will predominantly give rise to substitution at the ste cally least hindered position, i.e. the τ-position. This is in particular the case with sterically hindered reagents.

In Boc chemistry the dinitrophenyl group has been extensively employed but suffers from the disadvantage that it has to be removed in a separate step with thiophenol or other thiols in dimethylformamide (DMF).

The benzyloxymethyl (Bom) is a protective group on the π-nitrogen in Boc chemistry. This protective group can give rise to formaldehyde during the splitting off by acid, leading to the formation of several side products as shown by Mitchell et al in Int. J. Peptide Protein Res., 1990, 36, pages 350-355. The trityl group is widely used in Fmoc chemistry but the trityl group is not a , is not strongly electron-withdrawing protective group, and it is therefore not surprising that Harding et al showed on poster P025 at the 23rd European

Peptide Symposium 1994, Braga, Portugal that " FmocHis(tTrt)OH suffers from gross racemization..."Programm and Abstract" from said symposium.

It can be concluded that although many protective groups for the imidazole nucleus has been suggested, for both Boc and Fmoc chemistry, these protective groups suffers from several distinct disadvantages. An ideal type of protective group would be of electrowithdrawing, acid removable such as Boc or benzyloxycarbonyl groups, protective groups employed for the protection of primary and secondary amino groups.. Unfortunately these types of protective groups are not stable towards nucleophiles. The Boc group has been used but Sieber et al reported in Tetrahedron Letters vol. 28, No. 46, pages 6031-6034, that the Boc group was unstable to nucleophiles (see said report, page 6031 , lines 5 - 7, second section).

In a recent report Nishiyama et al , in J. Chem. Soc. Chem. Commun., 1994, pages 2515- 2516, suggested that the N^ι -2-adamantyloxycarbonyl group and the N^ιm-1-adamantyloxycarbonyl group are suitable as protective groups for histidine in Boc and Fmoc, respectively. These derivatives were moderately stable towards nucleophiles, and 45 and 7 %, respectively, was cleaved off during a 10 minutes treatment with 20% piperidine in dimethylformamide. Clearly 7% of cleavage is far to labile for Fmoc chemistry where piperidine is used for cleavage of the Fmoc group. In Boc chemistry piperidines are not employed during simple standard synthesis of peptides but the N-terminal of the peptide chain is nucleophilic, and the N^ιm to N^α transfer of a protective group is always a danger with nucleophile-sensitive protective groups. Thus, the nucleophile-sensitivity of the protective groups should be kept at as low a level as possible to reduce the risk of irreversible transfer the nucleophilic N- terminal.

The phenolic hydroxyl group in tyrosine was in Boc chemistry originally protected as a benzylether. The disadvantage of this group was the migration of the benzyl cation to the ortho position of tyrosine during acidolytic cleavage. Therefore, the 2-bromobenzyloxycarbonyl group (2-BrZ) is now a standard protective group. Studies has shown that it can be cleaved during prolonged treatment with diisopropylethylamine. John M. Stewart concluded in is extensive review entitled "Protection of the hydroxyl group in peptide synthesis" published in the series The Peptides vol. 3 (Gross E. and Meienhofer J. Eds, Academic Press) page 192 lines 24-30, that :

"Although O-2-bromobenzyloxycarbonyltyrosine is the best derivative currently available for solid phase peptide synthesis, some improvement may still be in order for the synthesis of very long peptides; because 24-hr treatment with trifluoroacetic acid in dichloromethan causes a 1% loss of the blocking group, while a similar treatment with 10% diisopropylethylamine in dimethylformamide caused a 5 % loss of the blocking group".

Another side reaction relevant in this context is that during the cleavage of the Boc group in Boc chemistry, the fe/ -butyl cation formed in said reaction can alkylate protected cysteine residues forming sulphonium salts. This reaction can be partly suppressed by the addition of thiol scavengers. However, this has disadvantages such as strong unpleasant smell and this strategy is not always effective. Protective groups that protected against said fert-butylation of the sulphur in cysteine would therefore be desirable. The protective groups used in the prior art as protective groups for the side chains in peptide synthesis has predominantly been the following:

1_. Aromatic protective groups such as benzylester, ethers or carbamates, . Cycloalkanes such as the cyclohexyl and cyclopentyl esters suggested as protective groups for aspartic and glutamic acid in Boc chemistry, and 2__The simple tertiary acyclic tert-butyl group in the form of ester, carbamate or ethers in Fmoc chemistry.

Acyclic alkyl protective groups that can be cleaved by anhydrous acid having more then 5 carbons in the alkyl group has apparently only been reported once, in 1957 by McKay and Albertson who reported in J. Am. Chem. Soc. Vol. LXXIX, pages 6180-6183, that the carbodiisopropylmethoxy group could be employed as a protective group for the C^α-amino group of glycine. In this case the carbodiisopropylmethoxy group was not used to protect a side chain of an amino acid but rather the N^α- amino group of glycine. The conclusion drawn from the experiments with this protective group by the authors were that : "Since slow removal of the urethan-protecting group allows a competing reaction to become of more importance work with the chloroformats of cyclohexanol and diisopropylcarbinol was discontinued when it became apparent that experimental results were bearing out the superiority of the carbocyclopentyloxy group". Said publication page 4688 , line 26-.

In summary, acyclic aliphatic protective groups has not prior to the present invention been considered to have any advantages in peptide synthesis. This is reflected in the fact that this type of protective groups has not been employed as protective groups for the side chains of amino acids.

Summary of the invention The present invention provides new protective groups bound to side chains of amino-acid moieties which do not have the disadvantages of the hitherto used protected derivatives in peptide synthesis. Protective groups bound to the side chains of amino-acid moieties, which groups are composed of acyclic saturated branched hydrocarbons with between 6 - 18 carbon atoms are more flexible than analogous cyclic structures. Protective groups of this type are able to protect the protected polypeptide chain or protein chain from side reactions during the synthesis. The new protective groups according to the invention have properties superior to protective groups previously reported. They are more stable during the synthesis or protects from modification to a significantly greater extent than previously reported comparable protective groups. They can be introduced into the peptide chain by standard procedures and can be cleaved with standard procedures for peptide synthesis.

Description of the invention The present invention is directed to a protective group bound to a side chain of an amino-acid moiety , which group is an aliphatic acyclic branched alkyl group protecting a thiol, hydroxyl, or carboxyl group or an aliphatic acyclic branched alkoxycarbonyl group protecting an amino group or heterocyclic nitrogen, wherein the alkyl and alkoxy entities have from 6 to 18 carbon atoms and have at least two identical alkyl chains.

An amino-acid moiety may in the present invention be a protected or deprotected form of an amino acid, a resin-bound amino-acid ,or an amino- acid residue forming part of a peptide, polypeptide or protein. For example, the amino-acid moiety may be protected so that the N^α amino group is protected by a te/ -butyloxycarbonyl group or 9-fluorenyl-methoxy-carbonyl group or benzyloxycarbonyl group, and the optionally modified carboxyl group is (i) present as an unmodified COOH group, (ii) modified by a protecting group (iii) modified by an activating group, or (iv) bound to the linker of a resin.

In an embodiment of the protective group of the invention the branched alkyl or alkoxy entity has no more than two identical alkyl chains.

In another embodiment the branched alkyl or alkoxy entity has three alkyl chains, at least two of which are identical.

In a preferred embodiment of the invention the amino-acid moiety derives from an amino acid which is selected from the group consisting of histidine, tryptophan, tyrosine and lysine, and said protecting group is an alkoxycarbonyl group.

In another preferred embodiment of the invention the amino-acid moiety derives from an amino acid which is selected from the group consisting of cysteine, serine, threonine, aspartic acid and glutamic acid and said protecting group is an alkyl group.

In a particularly preferred embodiment of the invention the alkyl entity of the protective group has 7 carbon atoms.

In another particularly preferred embodiment of the invention the alkyl entity of the protective group has 9 carbon atoms.

Another aspect of the invention is directed to the use of a protective group bound to a side chain of an amino-acid moiety in the synthesis of peptides, polypeptides and proteins. Thus, the aliphatic acyclic branched alkyl entities of the protective groups bound to side chains of amino-acid moieties according to the invention may form side-chain protected amino acids which, when used in peptide synthesis, have properties superior to currently used protective groups for side chains of amino acids. A protective group of the invention generally has larger flexibility than a cyclic aliphatic protective group or a protective group of aromatic character such as a benzyl ester.

Furthermore, an important aspect of such acyclic hydrocarbons is that they will provide sterical hindrance which may lead to an effect that certain interactions occur less frequently and thereby, if these interactions will lead to undesired side reactions, lower the amount of said side reactions. Several different classes of side reactions can be prevented or reduced: 1. Side reactions as a result of intramolecular interactions such as aspartimide formation.

2. Reactions with reactive molecules in the solvent. As an example of this is the nucleophile sensitivity of the urethan type of protective groups on the imidazole nucleus of histidine or the phenolic alcohol group on tyrosine. In the comparative examples given below is shown that acyclic alkyloxycarbonyl groups derived from secondary alcoholes gives superior protection against nucleophilic cleavage by a nucleophile such as piperidine. And importantly, the comparative examples clearly show that a very large difference in efficiency of the protection is recorded when comparing cyclic and acyclic aliphatic protective groups such as the ones shown in structural formulae (1) . It is noted that compared with the similar, branched 2-adamantyl ester which is a c^yclic aliphatic ester, the acyclic β-2,4-dimethyl-3-pentyl ester of aspartic acid gives far superior protection against base catalyzed intramolecular aspartimide formations and premature removal by nucleophiles. Branched in this context refers to the two methyl groups in position 2 and 4 and comparable position in 2-adamantol. Structural formulae (1)

II

Structure of a 2-adamantyl ester and (I) and a 2,4-dimethyl-3-pentyl ester (II)

For comparative examples, see figuresl &2 and Table 1.

These acyclic alkyl protective groups bound to the side chains can be used as protective groups in two chemical forms: 1. Alkyl groups where the alkyl entities are linked directly to the side chain of the amino acid. These include protective groups for the side chains of Ser, Thr and Cys (ethers) and Asp, Glu (esters)

2. Alkyl groups of the structures given in 1. above but where the alkyl entity and the side chain of the amino acid are linked to each other by a oxycarbonyl group, forming a alkyloxycarbonyl type of protective group, i.e.

alkyl group-O-CO- amino-acid side chain.

In this form new innovative protective groups for the side chains of His, Trp, Tyr and Lys are provided. In the case of histidine both the π and τ nitrogen can be employed for derivatization.

In several cases, two classes of protecive groups can be constructed from these principles.

A. If a secondary alkyl group is used, a protective group with an acid lability compatible with Boc chemistry (stable to trifluoroacetic acid, cleaved by hydrogen fluoride ) is obtained. If a tertiary alkyl group is used, a protective group with an acid lability compatible with Fmoc chemistry is obtained (stable to piperidine but cleaved with trifluoroacetic acid). An example of this is given in structural formulae (2) below. Structural formulae (2)

III IV VI

The histidine and tyrosine derivatives of III and V are new innovative nucleophile resistant protected derivatives where the protective group is stable to trifluoroacetic acid but readily cleaved with hydrogen fluoride (Boc chemistry). In contrast, similar use of the derivatives IV and VI will provide nucleophile resistant but trifluoroacetic acid labile protective groups for histidine in Fmoc chemistry. In the particual case of histidine, these protective groups suppress racemization, are cleaved by anhydrous acid and is not known to form any other harmful side products during the cleavage than the alkyl carbocation.

Another example can be put forward in the case of tyrosine. Here alkoxycarbonyl groups such as 2,4-dimethyl-3-pentyloxycarbonyl and 2,6- dimethyl-4-heptyloxycarbonyl groups in structural formulae (2) above can be used as nucleophile resistant, hydrogen fluoride cleavable protective groups. See comparative example 3 below.

In the case of tryptophan, similar protective groups as for tyrosine and histidine can be employed to protect the indole nucleus of tryptophane. Introduction of alkyloxycarbonyl groups as protective groups for tryptophane has been shown to protect the indole nucleus from alkylation. Alkyloxycarbonyl groups of a design similar to those suggesed for Tyr and His will provide that protection but these groups will be more resistant to premature cleavage by nucleophiles.

Introduction of flexible alkyl protective groups will also increase the solubilities of the peptides in organic solvents during the synthesis. This will most certainly be a big advantage as it will in some cases decrease the interaction between two or more peptide chains.

Protection of the thiolgroup of cysteine in the form of tertiary thioether by sterically hindered tertiary S-alkyl groups can reduce the rate of S-tert butylation of cysteine forming a sulphonium salt derivative during the removal of the Boc group in Boc chemistry.

The present invention can be be applied with a large number of different hydrocabons but the general idea is that they should not contain any cyclic hydrocarbon as these will provide less serical hindrance and flexibility. Displayed below are the structural formulae (3) comprising examples of alkyl entities of protective groups according to the invention.

Structural formulae (3) ϊ^> Examples of different new

R₁- C - R2 protective groups wherein both of R1 and R2

- CH₃ - CH₂CH₃ -CH₂(CH₂)_nCH₃ represent the same IX-XII

VII VIII K and R3 represents

CH_{3 y}CH₃ _CH₃

1 hydrogen or VII -XI,

-(CH₂)-C-CH₃ (CH₂)_n-CH -CH,

"n 1

CH₃ CH₃

CH₃ n =1-6.. X XI XII

R1 and R2 can be VII and R3 IX.X, XI wherein n = 1-13.

Description of the drawings

Fig 1 shows the effect of treatment of the model peptide: Boc-Lys(2CIZ)-

Tyr(2BrZ)-Asp(Xpp)-Gly-Phe linked to a p-methylbenzhydrylamine resin with 20% piperidine in dimethylformamide for four hours followed by cleavage of the protected peptide-resin with liquid hydrogen fluoride and chromatographical analysis of the product on a reversed phase HPLC column using a gradient of 80% acetonitrile 20% water. Xpp indicated different protective groups on the β-COOH group of aspartic acid. Aspartyl peptide indicate the the unmodified product Lys-Tyr-Asp-Gly-Phe-NH2. A. The commercial available cyclohexyl ester. B: The new innovative β-2,4-dimethyl-

3-pentyl ester. Fig. 2. shows the effect of treatment of the model peptide: Boc-Lys(2CIZ)- Tyr(2BrZ)-Asp(Xpp)-Gly-Phe, linked to a p-methylbenzhydryl-amine resin with 20% piperidine in dimethylformamide for fourteen hours followed by cleavage of the protected peptide-resin with liquid hydrogen fluoride and chromatographical analysis of the product on a reversed phase HPLC column using a gradient of 80% acetonitrile 20% water. Xpp indicated different protective groups on the β-COOH group of aspartic acid. Arrows indicate the the unmodified product Lys-Tyr-Asp-Gly-Phe-NH2 A: the new innovative β-2,4-dimethyl- 3-pentyl ester. B. β-Amenthyl ester. C. β- 2-adamantyl ester

EXAMPLES

As representative examples are given the preparation of an alkylester and alkyloxy carbonyl protective group designed according to these new innovative principes.

EXAMPLE 1

Synthesis of secondary alcohols

Secondary alcohols can be synthesized by well established procedures from alkyl halides and aldehydes by using the Grignard reaction. A description of such procedures can be found in Organic Synthesis collective volume 2, pages 406-407. Blatt A.H. Ed., John Wiley and Sons.

EXAMPLE 2

Synthesis of tertiary alcohols Tertiary alcohols can be synthesized by well established procedures from alkyl halides and ketones by using the Grignard reaction. A description of such procedures can be found in Vogel^'s textbook of practical organic chemistry, fifth edition, Furniss, B.S., Hannaford, A.J., Smith, P.W., Tatchell A.R., 1991 , ISBN 0-582-46236-3, Longman Scientific & Technical, pages 538-539.

EXAMPLE 3

Synthesis of chloroformates of secondary alcohols To one equivalent of secondary alcohol such as 2,4-dimethyl-3-pentanol or 2,6-dimethyl-4-heptanol is added 1.2 equivalents of phosgene (1.98 M solution in toluene). The solution is stirred at room temperature for 6 hours. Residual phosgene is removed by passing a stream of dry nitrogen through the solution. The resulting 2,4-dimethylpentyl-chloroformate and 2,6-dimethyl- 4-heptylchloroformate are used in the reactions described below as the toluene solutions obtained by this procedure.

EXAMPLE 4 Synthesis of fluoroformates of tertiary alcohols.

Since chloroformates or other similar type of activation of tertiary alcohols in order to prepare alkoxycarbonyl protective groups are difficult to achieve via procedures applicable to a secondary alcohol, activation of a tertiary alcohol can be achieved by preparing the fluoroformates of said alcohol. A procedure for the preparation of fluoroformates of a tertiary alcohol, which is used to introduce super acid labile N^α-protective groups for use in peptide syntheses, is described by Voelter and Mϋller in Leibigs Ann. Chem. ,1983, pages 248- 260.

EXAMPLE 5

Introduction of haloformates of secondary or tertiary alcohols to the imidazole nucleus of histidine.

One equivalent of N^α-Boc-Histidine is dissolved in dimethyl sulphoxide and two equivalents of diisopropylethylamine is added. To this solution is added 1 equivalent of chloroformates of secondary alcohols or fluoroformates of tertiary alcohols prepared as described in example 3 or 4. The solution is stirred for 10 minutes at room temperature. The reaction mixture is added to a twenty times larger volyme of ethyl acetate and washed with an equal volyme of water containing two equivalents of acetic acid. The aqueous phase is discarded and the ethyl acetate phase is washed once with water. The ethyl acetate phase is dried over magnesium sulphate, filtered and concentarated by evaporation. Aliquotes of the resulting oil is dissolved in a mixture of acetonitrile and water containing 0.1 % trifluroacetic acid and purified by High Performance Liquid Chromatography (HPLC) using a reversed phase column. The acetonitrile in fractions containing the product isolated by chromatography is evaporated, saturated sodium chloride and ethyl acetate is added, and the organic layer is washed two times with saturated sodium chloride solution. After drying over MgS0₄the solvent is evaporated. The product is dried over night in high vaccum, re dissolved in hexane and the solvent evaporated. This procedure was repeated four times and the resulting oil dried overnight.

EXAMPLE 6 Esterification of the β-COOH group of Nⁿ-Boc-Asp-OBzl

One equivalent of Boc-Asp-OBzl is dissolved in methylene chloride and three equivalents of 2,5-dimethyl-3-pentanol is added followed by 0.5 equivalents of dicyclohexylcarbodiimide. The solution is stirred for 15 minutes and 0.05 equivalents of N,N-dimethylaminopyridine is added and the reaction mixture is stirred for 3 hours. After this period of time, additional 0,5 equivalents of dicyclohexylcarbodiimide is added and the reaction mixture is stirred for 16 hours. The precipitated dicyclohexylurea is removed by filtration and the methylene chloride is removed by evaporation. The resulting oil is re dissolved in ethyl acetate and is washed with 0.1 M HCI (3 times), 0.1 M sodium hydrogen carbonate (3 times) and saturated sodium chloride solution (3 times).The ethyl acetate solution was dried with MgS0₄, filtered and an equal volume of hexane was added. This solution was filtered through a funnel containing silica to remove remaining traces of dicyclohexylurea followed by evaporation of the solvents. The resulting oil was re dissolved in ethanol, followed by evaporation of the solvent in order to remove residual ethyl acetate. This procedure was repeated twice. The resulting oil containing an aspartic acid derivative wherein the N amino group is protected by the Boc group, the α-COOH protected as a benzyl ester and the β-COOH group protected as a 2,4-dimethyl-3-pentyl ester was obtained as an oil which was used without further purification in the next step of synthesis (example 7).

EXAMPLE 7 Splitting off of the benzyl-protective group on the C" COOH group of the aspartic acid derivative obtained in example 7.

One equivalent of the intermediate product obtained in example 7 was dissolved in ethanol and a hydrogenating catalyst, 5% palladium on charcoal, was added. The said catalyst was added in an amount of 1 part by weight per 20 parts by weight of the starting material. The reaction mixture is flushed by nitrogen and hydrogen gas is bubbled through the said mixture for 4 hours. The reaction mixture is flushed by nitrogen, transferred to a centrifugation tube and centrifuged so that the catalyst is obtained as a pellet at the bottom of the centrifugation tube. The supernatant is collected, the solvent is removed by evaporation, the resulting oil is re dissolved in ethyl acetate, and the product is shaken into an aqueous solution of sodium bicarbonate. This solution is washed with diethylether (3 times) followed by acidification with solid citric acid and addition of diethyl ether. The organic phase is washed with saturated sodium chloride, dried over magnesium sulphate, and the solvent is evaporated. The resulting oil is re dissolved in hexane and the hexane is evaporated. This procedure is repeated twice. After removal of residual diethyl ether the desired derivative N^αBoc-Asp(2,4-Dimethyl-3-pentyl)OH can be crystallised from warm hexane and the product is obtained as crystals.

EXAMPLE 8

Preparation of a new innovative protected derivatives of tyrosine.

The chloroformates were prepared according to example 3. One equivalent Boc-Tyr-OH was dissolved in acetonitrile, 1.1 equivalent N,N diisopropyl-ethyl amine was added followed by 1 equivalent benzyl bromide. After 24 hours, the solvent was evaporated, the resulting oil taken up in ethyl acetate and washed with 1 N HCI (3 times) followed by water (3 times) . The product was shaken into a water solution of 1.1 equivalent NaOH, followed by acidification with solid citric acid and the Boc-Tyr-OBzl was taken up in ethyl acetate. The product was crystallised from ethyl acetate and hexane. The crystals were dissolved in tetrahydrofuran, 1 equivalents of sodium hydride was added followed by 1.1 equivalents of the alkylchloroformate described in example 3.

Five equivalents of acetic acid was added and the solvent removed by evaporation. The resulting oil is re dissolved in ethyl acetate and is washed with 0.1 M HCI (3 times), 0.1 M sodium hydrogen carbonate (3 times) and saturated sodium chloride solution (3 times). The resulting oil was re dissolved in ethanol, followed by evaporation of the solvent in order to remove residual ethyl acetate. This procedure was repeated twice.

This product where the phenolic hydroxyl group is protected as the derivative A&B in table 2 was subjected to catalytic hydrogenation as in example 7 in order to remove the benzyl ester protective group on the oc-COOH group. The procedure after this step was the same as described in said example 8. The product is obtained as an oil.

EXAMPLE 9

Preparation of a polypeptide by using the protected aspartic acid derivative of example 7

The polypeptide of the sequences stated above was prepared by solid phase peptide synthesis using conventional Boc chemistry.. A p- methylbenzhydrylamine resin containing 1 equivalent of amino groups was washed with 2x5% N,N-diisopropylethylamine in methylene chloride and washed 4 times with methylene chloride. After these washes the resin was resuspended in a small volyme of methylene chloride. The first Boc amino acid was activated by mixing 1 equivalent of Boc amino acid, 1.1 equivalents of N-hydroxybenzotriazole and 1 equivalent of dicyclohexylcarbodiimide in N,N-dimethylformamide (DMF). After 15 minutes the reaction mixture was transferred to the said resin and the reaction mixture was shaken for 30 minutes. After this period of time, the solvents were aspirated and the resin was washed with absolute ethanol (three times) , DMF (three times) and methylene chloride (5 times). A solution of 50 % trifluoroacetic acid in methylene chloride was added and the reaction was shaken for 1 minute, the solvents aspirated and the procedure repeated once for 18 minutes. The resin was aspirated and washed with methylene chloride

(three times) and absolute ethanol (three times) , 5 % N,N- diisopropyl¬ ethylamine in DMF (two times) and metylene chloride (four times). After aspiration of the metylene chloride added in the final washing step the free amino groups on the resin were acylated by the second Boc amino acid activated by the said procedure.

The procedures for cleavage of the Boc group, washing and acylation were repeated until a protected resin-bound polypeptide of the desired sequence was synthesized. Final deprotection was achieved by treating 100 mg of the peptide resin with 5 ml liquid hydrogen fluoride containing 0.5 grams of p- cresol at 0°C for 40 minutes. The hydrogen fluoride was evaporated, diethyl ether was added and the precipitated peptide was collected by filtration. Water was added to the precipitated product, the solution was filtered and the filtrate was lyophilised. The resulting powder was analysed and purified on PLC using a reversed phase column. The peptides were characterised by plasma desorption mass spectrometry using a time of flight detector.

COMPARATIVE EXAMPLE 1

The β-2,4-dimethyl-3-pentyl ester of aspartic acid compared with other protective groups reported in the literature.

The usefulness of the invention is exemplified by experimental studies described below.

A peptide of the following sequence was synthesized: Boc- Lys(2-CI-Z)-Tyr(2BrZ)- Asp(Xpp)-Gly-Phe-MBHA, where Xpp denotes the aspartic acid protective groups studied, and MBHA the p-methylbenzhydryl amine resin. Xpp can be cyclohexyl or 2,4-dimethyl-3-pentyl. The protected peptide resin was treated with 20% piperidine in DMF for 4 hours, cleaved in liquid hydrogen fluoride and the purity of the product assayed by HPLC using a reversed phase column. The elution profiles from these experiments are shown in fig .1. A. cyclohexyl protection, B 2,4-dimethyl-3-pentyl protection. As it is well-known that hydrogen fluoride treatment in it self causes a few % acid catalyzed aspartimid formation, the traces of impurities in B where the new innovative protective group is used, does only partly arise from base catalyzed aspartimide formation. Thus the superiority of the new innovative protective group is even greater then indicated in figure 2. The benzyl and cyclohexyl esters are standard protective groups in Boc chemistry for aspartic acid. The benzyl ester was not included in the study as it is well known that this type of protection results in even higher levels of aspartimide under these reaction conditions.

The main product in B denoted aspartyl peptide is the correct product as indicated by plasma desorbtion mass spectrometry and reference peptides prepared without piperidine treatment.

The superiority of this new innovative protective group is further supported by in studies where it is compared with the aspartic acid residues protected at the β-COOH group as β-mentenyl esters or β-2-adamantyl esters.. The said model peptide; Boc-Lys(2-CI-Z)-Tyr(2BrZ)- Asp(Xpp)-Gly-Phe- linked to a p- methylbenzhydrylamine resin was prepared but in this case the protected peptide was treated with 20% piperidin in DMF for 14 hours. Here it is clearly demonstrated that acyclic structures are superior to comparable cyclic structures ( se structural formula 1 above) in preventing base catalyzed aspartimide formation. In both the examples given above all protecting groups gave clean homogenous products if the aspartic acid derivatives were incorporated into the said peptide and no piperidine treatment was carried out and the protected peptide treated with hydrogen fluoride. The only significant side products were the said low levels of acid catalyzed aspartimide. The acid stability of the 2,4 dimethyl-3-pentyl ester as compared with the commercial available β-benzyl, β-cyciohexyl esters of aspartic acid was investigated and a acid stability greater then the benzyl ester but more labile then the cyclohexyl ester was obtained. As these two commercial available esters of aspartic acid both has acid stability that makes then suitable for peptide synthesis with Boc chemistry is it can be concluded that the acid stability of the innovative 2,4 dimethyl-3- pentyl ester is sufficient stable for trifluoroacetic acid but can be cleaved by liquid hydrogen fluoride. COMPARATIVE EXAMPLE 2

Acyclic alkoxycarbonyl derivatives of histidine as nucleofile resistent protective groups.

Protecting of the imidazole nucleus of histidine with uretan type of protective groups has been very little employed as these protective groups are readely removed by nucleofiles. By introducing the 2,4 dimetyl-3-pentyloxycarbonyl or the 2,6 dimetylheptyloxycarbonyl groups as protective groups for the imidazol ring of histidine proteceted derivates are obtained that shows a large stabiliy against nucleofiles. In table 1 these new innovative derivates of histine are compared with other urethan type of protective groups such as the isopropyloxycarbonyl and the 2-adamantyl and 1 -adamantyloxycarbonyl derivates reported by Nishiyama et al 1994. Table 1

N"" protected dervates of histidine Relative amount of protective group cleaved by 20% piperidine

A. Isopropyloxycarbonyl t _V2 less them 5 minutes

B. 2,4-Dimethyl-3-pentyloxycarbonyl 20% at 24 hours

C. 2,6-Dimethyl-4-heptyloxycarbonyl /₂ » 7 hours

D. 2-Adamantyloxycarbonyl 45% in 10 minutes ##

E. 1 -Adamantyloxycarbonyl 7% 10 minutes ^{## DDD}

## Nishiyama et al 1994

ODD derivative of a tertiary alcohol

The model peptide Boc-His(Xpp)-Ala-Pro-Lys(Boc)-Tyr(tBu) where Xpp indicate the protective groups displayed in table 1 , was synthetized on an p- methylbenzhydrylamine resin to which a trifluoroacetic acid labile trialkoxybenzhydrylamine linker was coupled by dicyclohexylcarbodiimide. Peptide synthesis was carried out with Fmoc amino acid which were activated by addition of 1.0 equivalent dicyclohexylcarbodiimide to a solution of Fmoc amino acid and 1 ,1 equivalent N-hydroxybenzotriazol (HOBt) forming a so called HOBt ester. Couplings were carried out in DMF. Cleavage of the Fmoc group was performed by 20% piperidine in DMF. The N-terminal N^im protected histidine derivative was introduced protected at the N^α-amino group with the

Boc group. These Boc-His(Xpp)OH amino acid derivatives, where Xpp denotes the protective groups A-C in table 1 , was coupled with the BOP reagent. The peptides were treated with 20 % piperidine in DMF, the peptides cleaved from the resins by trifluoroacetic acid and the ratio between peptides with protective group on the histidine residue and peptides were the protective group has been removed by the action of piperidine was recorded by analysing the HPLC elution profiles and identification of the products by plasma desorbtion mass spectrometry. For comparison the stability of the N^im- 2- adamantyl and N^im-1-adamantyl derivatives of histidine as reported by

Nishiyama et al 1994 is included. If these protected peptides were treated with liquid hydrogen fluoride identical homogenous products were obtained. It is clear from this study that the derivatives A,B,C & D, that all are compatible with Boc chemistry, the Nⁱ protective group is cleaved with liquid hydrogen fluoride, but importantly, the new innovative derivatives B & C designed from the said new innovative principle is superior to the derivative D and even more nucleophile resistant then the derivative E which is alkoxycarbonyl derivative of a tertiary alcohol which by its structure in itself is more sterical hindered then a comparable derivative of a secondary alcohol.

COMPARATIVE EXAMPLE 3

Acyclic alkoxycarbonyl derivatives of tyrosine as nucleophile resistant protective groups.

As refereed to above, the 2-Br benzyloxycarbonyl protective group is the standard protective group for the phenolic OH group of tyrosine in Boc chemistry , but that this protective group has some disadvantages in terms of nucleophile sensitivity. By introducing the 2,4-dimethyl-3-pentyloxycarbonyl or the 2,6-dimethyl-4-heptyloxycarbonyl groups as protective groups for phenolic OH group on tyrosine O-protected derivatives of tyrosine are obtained that shows a large stability against nucleophiles. In table on page 2 these new innovative derivatives of histidine are compared with the 2-Br benzyloxycarbonyl protective group. The model peptide Boc-Tyr(Xpp)-Ala-Pro- Lys(Boc)-Tyr(tBu) was synthesised on an p-methylbenzhydrylamine resin to which a trifluoroacetic acid labile trialkoxybenzhydrylamine linker was coupled by dicyclohexylcarbodiimide. Peptide synthesis was carried out with Fmoc chemistry as describe above in comparative example 2. The last amino acid residue BocTyr(Xpp)OH, where Xpp denotes the protective groups A-C in table 2, was coupled as HOBt esters. The peptide resin was treated with 20% piperidine in DMF, cleaved with trifluoroacetic acid and the products analysed and purified as described in the comparative example 2 above. As trifluoroacetic acid cleaves all protective groups except the histidine protective groups, the relative amount of nucleophile cleaved protective groups can be recorded.

If the same peptide resin is treated with liquid hydrogen fluoride the protective groups shown in table 2 the protective groups A-C are cleaved and clean homogenous products are obtained.

table 2

O-protected derivatives of tyrosine Relative amount of protective group cleaved by 20% piperidine

A. 2,4-Dimethyl-2-pe ntyloxycarbonyl 20% at 24 hours

B. 2,6-Dimethyl-4-heptyloxycarbonyl t_{1 2} =7 hours

C. 2-Br benzyloxycarbonyl 100% at 5 minutes

A conclusion that can be drawn from table 2 is that the derivatives A & B has a nucleophile resistance that are orders of magnitude larger then the standard protective group for the side chain of tyrosine in Boc chemistry (C table 2).

Claims

1. Protective group bound to a side chain of an amino-acid moiety , c h a r a c t e r i z e d in that said group is an aliphatic acyclic branched alkyl group protecting a thiol, hydroxyl, or carboxyl group or an aliphatic acyclic branched alkoxycarbonyl group protecting an amino group or heterocyclic nitrogen, wherein the alkyl and alkoxy entities have from 6 to 18 carbon atoms and have at least two identical alkyl chains.

2. Protective group according to claim 1 , wherein said branched alkyl or alkoxy entity has no more than two identical alkyl chains.

3. Protective group according to claim 1 , wherein said branched alkyl or alkoxy entity has three alkyl chains, at least two of which are identical.

4. Protective group according to any one of claims 1-3, wherein said amino-acid moiety derives from an amino acid which is selected from the group consisting of histidine, tryptophan and lysine, and said protecting group is an alkoxycarbonyl group.

5. Protective group according to any one of claims 1 -3, wherein said amino- acid moiety derives from an amino acid which is selected from the group consisting of cysteine, serine, threonine, tyrosine, aspartic acid and glutamic acid and said protecting group is an alkyl group.

6. Protective group according to any one of claims 1-5, wherein said alkyl entity of the protective group has 7 carbon atoms.

7. Protective group according to any one of claims 1-5, wherein said alkyl entity of the protective group has 9 carbon atoms.

8. Use of a protective group bound to a side chain of an amino-acid moiety according to any one of the claims 1-7 in the synthesis of peptides, polypeptides and proteins.