HK1113376A - Cyclization of a peptide - Google Patents

Description

Cyclization of peptides

Technical Field

The present invention relates to improved methods of disulfide-bond driven peptide cyclization.

Background

Vapreotide (Vapreotide) is a cyclic octapeptide containing a cystine moiety. It is a synthetic analogue of an inhibitor of growth hormone release, which inhibits secretion of hormones such as insulin and glucagon (glucagon), and is an important regulator of obesity and carbohydrate metabolism and growth hormone. The physiological half-life of growth hormone release inhibitor is 3 minutes, while vapreotide maintains a longer circulation due to the C-terminal amidation of the analog. Disulfide bonds are important parameters for the activity of vapreotide and inhibitors of growth hormone release.

Growth hormone release inhibitors have the following structure:

vapreotide has the following structure:

Volkmer-Engert et al (Surface-assisted catalysis of intramolecular disulfide bond formation in peptides), J.peptide Res.51, 1998, 365-. Careful control indicates that physical dissolution of oxygen in aqueous media can be necessary and sufficient to afford oxidation of the char with oxygen. The use of carbon significantly and selectively accelerates the rate of the intramolecular cystine formation reaction compared to conventional gas stream sparging in the absence of a catalyst.

The use of carbon as a heterogeneous catalyst inevitably requires that the reaction be carried out in solution rather than on the resin. Has become a widely applicable method in the applicable field; however, some of the limitations of this approach to solvent systems are not applicable to every peptide.

US6476186 proposes intramolecular disulphide bonding of linear deprotected octapeptide D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr (OH) in acetonitrile/water (1: 1) in the presence of trace amounts of charcoal. This peptide was synthesized on 2-chlorotrityl resin and contains lysine and threonine in addition to hydrophobic residues and cysteine. Cysteine is protected with an acid-labile trityl group. The carbon-catalyzed cyclization was carried out after cleavage and deprotection in an aqueous solvent mixture, carefully adjusted to pH 8.0 with sodium hydroxide at a concentration of 50mg/mL for 5 hours. The yield of pure product was about 80%.

There is a drawback that when seeking to apply the same method to non-cyclized vapreotide, a peptidamide (peptamide) of low water solubility, 80% analytical yield could be achieved only with extended reaction times of 44 hours and at about 1mg/mL dilute solution. The concentration rises only to 5-10mg/mL and a slow precipitation of the educt occurs, thus reducing the product yield. By changing the pH to 6, a high concentration of about 10mg/mL can be achieved, but by-products are also formed, thus reducing the yield to 30%. Shorter reaction times and higher yields are required to achieve an efficient industrial scale process.

Disclosure of Invention

It is an object of the present invention to provide an alternative or improved process for cyclising a linear peptide precursor of mature vapreotide, which avoids the drawbacks of the prior art. The object of the invention is achieved by a process for cyclising a linear peptide (H) -D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp (NH2) by formation of intramolecular cystine, comprising the step of cyclising said peptide in a polar aprotic solvent at a concentration of at least 20mg/mL, in the presence of charcoal, in addition to a primary, secondary or tertiary amine as a basic reagent and an oxidising agent.

The peptides may be prepared by any method known in the art, may be prepared using biotechnology, or may be prepared by liquid or solid phase chemical synthesis. Solid phase Synthesis methods are described, for example, in Bodansky, M., Principles of Peptide Synthesis (Principles of Peptide Synthesis), 2 nd edition Berlin Springer Verlag/Heidelberg, 1993. C-terminal amidation can be performed by enzymatic or chemical methods known in the art, such as solid phase on resin synthesis by C-terminal ligation on well-known Sieber or Rink amide resins, yielding carboxamides (carboxamid) upon cleavage from the resin. Chemical synthesis typically requires the use of protecting groups for each amino acid, as is conventionally understood in the art. The residue of cysteine can be protected using various protecting groups, such as trityl, acetamidomethyl- (acm-), tert-butyl, trimethylacetamidomethyl, 2, 4, 6-trimethoxybenzyl, methoxytrityl, tert-butylsulfinyl (sulfophenyl). Removal of such protecting groups requires specific reagents or reaction conditions, depending on the type of protecting group used.

Trityl is most commonly used to provide simple protection in the synthesis of peptides, and this protecting group is then removed by simple acid hydrolysis, such as in 30-60% TFA, with the simultaneous removal of other protecting groups and resin linkers. For the purposes of the present invention, it is only necessary that the cysteinyl peptide (cysteinylpeptide) to be cyclized used in the process of the present invention has a free unprotected cysteine. Atherton et al (1985, j. chem. perkin trans. i., 2065) may be helpful as reported by the branch problem: the use of the well-known scavenger phenylthiomethane, which is a dual function of both scavenger and acidolysis promoter, also leads to partial deprotection of acm, t-butyl and S-t-butylsulfinyl protected cysteines upon acidolysis. This enables the invention to function. The S-tert-butyl-sulfinyl group can be easily and selectively removed under mild conditions by treatment with a thiol reagent such as β -mercapto-ethanol or dithio-threitol (threitol), or treatment with e.g. triphenylphosphine or triethylphosphine.

In contrast, in the art, acm-protected groups are typically removed by iodoxidation with simultaneous cyclization; thus, deprotection of the acm group supported on iodonium oxidation is not compatible with the present invention.

The polar aprotic solvent is preferably a solvent miscible with water and acetone. Suitable examples of solvents of this type according to the invention are, for example, Dimethylformamide (DMF), N-methyl-pyrrolidone (NMP), acetamide or Tetrahydrofuran (THF). Needless to say, a suitable solvent herein must be capable of achieving a peptide concentration of at least 20 mg/mL. Therefore, acetonitrile is not a suitable solvent for the present invention, as shown in the comparative examples of the experimental section.

The solvent is preferably selected from the group consisting of: acetamide, dimethylformamide and N-methyl-pyrrolidone. More preferably, the solvent is selected from dimethylformamide and N-methyl-pyrrolidone. The most preferred solvent is dimethylformamide.

The base is preferably a weak base, having a conjugate pKa of 7.5 to 10, and is a secondary amine, more preferably a sterically hindered tertiary amine. More preferred examples are Hunig-base (N, N-diisopropylethylamine), N' -dialkylaniline, 2, 4, 6-trialkylpyridine or N-alkyl-morpholine, the alkyl group being a linear or branched C1-C4 alkyl group, more preferably N-methylmorpholine or trimethylpyridine (2, 4, 6-trimethylpyridine), most preferably trimethylpyridine.

The oxidant is preferably air and/or oxygen. Changing from the prior art aqueous solvent system to the non-aqueous solvent system of the present invention, it is preferable to be able to ensure saturation of the catalyst with oxygen, at least for a certain period of time, by actively feeding or by ventilating the solvent liquid with air and/or oxygen by leading a steady or time-varying air flow from a gas shower immersed in the solvent. Optionally, a stirrer or stirring device with a possibly suitable shape, such as paddle or bead shape, can be used, with the strong vortex generated by stirring and the preferred aspect ratio of the liquid phase with respect to the vortex, to avoid only local mixing.

The carbon or activated carbon may be of the same type as the systems used in the prior art; preferably at least a catalytic amount is used. Needless to say, the reaction rate is influenced both by the amount of catalyst present and by the concentration of the educt, the latter increasing the rate or amount of adsorption or loading of the cysteinyl-peptide from solution on the solid surface of the heterogeneous catalyst. However, according to the present invention, the choice of solvent has a great influence on effective catalysis. Furthermore, it was observed (without wishing to be seriously affected by these observations or any relevant theory) that the reaction kinetics not only depend on the solvent system, but also are related to the amount of educts: when the amount of educt is increased to the range from 1 to 100mg/mL, the reaction kinetics are completely surprising changed in shape and are more linear than sigmoidal. Some transition phenomena at the solid-liquid phase boundary, which have not yet been reported, may account for this. As a result, the 100% conversion is somewhat equivalent to the increased product yield that can be achieved in a significantly shorter reaction time by increasing the concentration of educt. In the past, the use of carbon catalyzed oxidation reactions has not been demonstrated. Thus, according to the invention, at least 20mg/mL, preferably 50mg/mL, more preferably 100mg/mL of the cysteinyl peptide-precursor (H) -D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp (NH2) can be added to the reaction. The upper limit is of course determined by the maximum solubility.

The cyclization reaction can be carried out under reflux conditions at a temperature in the range of about 0 to 80 ℃, preferably 5 to 60 ℃, more preferably 15 to 40 ℃, and preferably in combination with air/oxygen as the oxidizing agent.

Experiment of

1. Synthesis of (H) -D-Phe-Cys-Tyr-D-Trp-Lys-VaI-Cys-Trp (NH2)

Linear peptides were synthesized on Sieber resin using Fmoc standard methods. Side chain protecting groups: D-oderL-Trp (Boc), Cys (Trt), Lys (Boc), Tyr (tBu). The protected peptide was cleaved in 5% TFA in dichloromethane and then deprotected completely by acid hydrolysis in a 300eq. (eq. parts by volume) concentrated TFA, 12 eq.dithiothreitol, 12 eq.dichloromethane, 50 eq.water (acqua dest.) cleavage mixture for 1 hour at room temperature. The product was precipitated by adding methyl-tert-butyl ether to completely remove the Boc group in the product (H) -D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp (NH2), and the purity was confirmed by HPLC.

All cyclization reactions were carried out at 25 ℃. The vapreotide product of formula I below can be purified by adding the reaction mixture directly to reverse phase HPLC.

2. Comparative example: (H) cyclization of-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp (NH2)

The product of experiment 1 was charcoal-treated essentially as described in US6476186, using acetonitrile: water (1: 1) except that the linear cysteinyl peptide concentration was 1mg/mL, the pH was adjusted to about 8 with ammonia, and the liquid was re-sprayed with low pressure air with stirring. After 44 hours, 100% conversion was achieved and the analytical yield was 84% (w/w) after purification of the product from the reaction mixture by fast HPLC. It was found in the past that higher concentrations of up to 10mg/mL could be unstable and the educts precipitated after a short time. By increasing the acetonitrile to water ratio to 3-2: 1, the peptide solubility was not significantly improved yet, while the reaction time at 1mg/mL was increased by 50-100% while the purity was reduced (yield: 74% (w/w)).

3. Comparative example: (H) cyclization of-D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp (NH2)

The reaction of example 2 was repeated, the only difference being the use of ammonia/NH₄Cl to adjust the pH to 6 and dissolve the cysteinyl peptide to 10 mg/mL. After 70 hours, the conversion reached 80%, but the product purity was lower. The yield of product I was 31%.

Cyclization of (H) -D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp (NH2)

The product of experiment 1 was subjected to the char method using a trace amount of activated powdered char, with a linear cysteinyl peptide concentration of 50mg/mL (1eq.) in dimethylformamide in the presence of 1eq.

After 15-20 hours, 100% conversion was achieved and the analytical yield of product I was 79%. The experiment was performed three times with the same results.

Claims (8)

1. A method for cyclising a linear peptide (H) -D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp (NH2) by formation of an intramolecular cystine, the method comprising the steps of: cyclizing the peptide in a polar aprotic solvent in the presence of carbon and in the presence of a primary, secondary or tertiary amine as a basic reagent and an oxidizing agent at a concentration of at least 20 mg/mL.

2. The method of claim 1, wherein the carbon is present in at least a catalytic amount.

3. The method of claim 1, wherein the oxidant is air and/or oxygen.

4. The method of claim 3, wherein air and/or oxygen is provided to the reaction during the reaction.

5. The process according to claim 4, wherein the reactor has means for introducing air and/or oxygen directly into the liquid, preferably comprising a plurality of holes in the bottom and/or walls of the reactor, or comprising at least one submerged gas sparger, through which air is bubbled directly into the solvent.

6. The method of any one of the preceding claims, wherein the polar aprotic solvent is a solvent selected from the group consisting of: a solvent miscible with both water and acetone, preferably selected from dimethylformamide or N-methyl-pyrrolidone.

7. The method of claim 6, wherein the solvent is dimethylformamide.

8. The method of claim 1, wherein the peptide is at a concentration of at least 50mg/mL, preferably wherein the solvent is dimethylformamide and the peptide is at a concentration of at least 100 mg/mL.