DD296083B5 - Process for the preparation of iodinated peptides and polypeptides - Google Patents

Description

H ₂ N-AS ₁ AS _n -B II

A-HN-AS ₁ AS _x AS _n -B III

2. The method according to claim 1, characterized in that the reaction of peptides / polypeptides which contain oxidation-sensitive amino acids in the association takes place with the ester of formula I.

The invention relates to a process for the preparation of iodinated peptides and polypeptides, wherein the peptide chains to be iodinated possess free amino groups at the N-terminus or in the side chain. The fields of application of the invention are the pharmaceutical industry and medical diagnostics.

During the usual direct iodination of peptides and polypeptides, e.g. With iodine monochloride (Mc Farlane, AS Nature, 190, 53 [1987]), chloramine T (Greenwood, F.C., Hunter, WM, Biochem J. 89, 114 [1963]), sodium hypochlorite (Redshaw, MR, Lynch. 5 BBRC 65 413 [1975]), the lodo gene method (Traker, PJ, Speck, J.C., BBRC 80 849 [1978]) and also in the iodination with lactoperoxidase / H ₂ O ₂ (Brundish, DE, Martin, JR, J., formerly Perkin I, 1976, 2182), the amino acids are exposed to strong oxidizing agents. Despite short reaction times, peptides with oxidation-sensitive amino acids such as methionine, tryptophan, cysteine, etc., have numerous by-products. The iodinated peptide is separated after extensive purification operations from the starting material and the by-products and isolated only in low yield. For the iodination of synthetic peptides, the use of iodinated amino acids is conceivable (eg, iodotyrosine, Brundish, DE, Wade, U., Biochem J. 165, 169 [1977]). The disadvantage of these syntheses, however, is the high synthesis and purification costs.

By linking the 4-hydroxy-5-iodo-phenylpropionic acid via the N-hydroxysuccinimide ester with the free amino acid of a peptide, iodine can subsequently be readily introduced into peptides (Bolton, AE, Hunter, WM, Biochem J. 133 529 [1973] ). The iodinated peptide is separable from the starting products by column chromatography.

A disadvantage of the use of this method is, above all, the low stability of the hydrosuccinimide ester of p-hydroxyphenylpropionic acid.

DE-OS 2 832 090 has reported the reaction of N (4-hydroxy-phenylethylcarbonyloxy) -norbom-5-ene-2,3-dicarboximide with peptides and the iodination of acyl peptides with chloramine-T and potassium iodide.

The reaction of N (4-hydroxy-5-iodo-phenyl-ethylcarbonyloxy) -norborn-5-ene-2,3-dicarboximide with peptides has not previously been described.

It is the object of the invention to provide iodinated peptides and polypeptides to the pharmaceutical and medical diagnostics which can be prepared by a favorable process.

The invention has for its object to develop a method by which peptides and polypeptides can be converted into their iodine derivatives. The object is achieved in that free amino-containing peptides or polypeptides of the general formulas II or III with N (4-hydroxy-5-iodo-phenyl-ethycarbonyloxy) -norbom-5-ene-2,3-dicarboximide of the formula I, in which I is also iodine ¹²⁵ , or reacted with N (4-hydroxy-phenyl-ethylcarbonyloxy) -norborn-5-ene-2,3-dicarboximide of the formula IV acylated and the acyl peptides or polypeptides are then iodinated.

A-HhMS ₁ - -A V ...

The peptide chains to be iodinated may consist of any amino acid residues and the amino groups to be acylated at the N-terminus or in the side chain. In the compounds of the general formulas II or III, the amino acid residues AS ₁ to AS _n can be any residues and AS _x an amino acid with an NH ₂ group in the side chain (eg lysine) and A and B in peptide chemistry also be other organic radicals. The peptide chains may also contain cysteine, tyrosine, methionine, histidine and also tryptophan.

For the acylation of the amino groups, an activated ester of p-hydroxyphenylpropionic acid was prepared, which is obtained in good yield and high purity without great expense, is stable in storage and can be reacted in good yields during the acylation reaction (formula IV). For the subsequent iodination of peptides with oxidation-sensitive amino acids in the peptide bond (eg methionine, tryptophan and cysteine), IV is converted to the monoiodo derivative I with chloramine T and potassium iodide prior to attachment to the free amino group of the peptide, chromatographically by HPLC on RP 18 of separated as the by-product diiodo product and reacted with the peptide to the iodine derivative quantitatively in 2 to 3-fold excess. By this procedure hardly any loss of peptide occurs.

Surprisingly, the following effects were observed: The corresponding ONB esters are more stable in aqueous solution than the hydroxysuccinimide esters and give fewer by-products. The hydrolytic stability of the ONB esters is increased by a factor of 10 compared to the known hydroxysuccinimide esters. In addition, unlike the hydroxysuccinimide esters, they are indefinitely stable at room temperature.

If no oxidation-sensitive amino acids are present in the peptide dressing, then the peptide can only be acylated with the N-hydroxy-norborn-S-ene-S-dicarboximide ester of the hydroxyphenylpropionic acid formula IV according to the abovementioned procedure and then with chloramine T and potassium iodide or K ¹²⁵ to afford the iodine derivative be modified. The separation of the by-produced diiodo derivative succeeds without problems on the final purification by HPLC on RP 18.

applications

Example 1: N (4-Hydroxy-phenyl-ethylcarbonyloxy) -norborn-5-ene-2,3-dicarboximide III 1.66 g (10 mmol) of hydroxyphenylpropionic acid and 1.79 g (10 mmol) of N-hydroxy-norbornene S-en ^ .S-dicarboximide (HON) are dissolved in ml of tetrahydrofuran (THF), cooled to 0 "C, with 2.475 g (12 mmol) of dicyclohexylcarbodiimide, and placed in the refrigerator over power. the mixture is diluted with 2 drops The resulting urea is separated off and washed twice with 2 ml portions of THF twice, and the purified filtrates are concentrated.

Resulting urea is previously separated. Ether is added to the resulting oil, concentrated again and the resulting solid is recrystallized from ethanol.

Yield: 2.9 g 88.6%

white crystals mp .: 141-142 °

M ^ -C ₁₈ H ₁₇ NO ₅ 327,1108 Characterization of the substance by the molecular ion peak in mass spectroscopy

Example 2: N (4-Hydroxy-5-iodo-phenyl-ethylcarbonyloxy) -norbom-5-ene-2,3-dicarboximide IV To 32 mg (97.8 μΜοί) of N (4-hydroxy-phenylethylcarbonyloxy) -norbornene 5-ene-2,3-dicarboximide III in 500 μΐ dioxane is added 9 mg (54 μΜοί) of potassium iodide in 200 μΙ sodium acetate buffer pH 7.5 (0.25 molar) containing 11 mg (48.9 μΜοί) of chloramine T in 500 μΐ Ammoniumacetatpuffer added and stirred for 30 see. The reaction is stopped with 3.7 g (24 μΜοί) of dithioerithritol, the clear reaction solution is injected into the HPLC and the corresponding fraction is separated off after chromatography on Vydac RP 18.

Yield: 10 mg 46% (after two lyophilizations)

M * .C ₈ H ₁₆ NO ₅ 453,0079 Characterization by mass spectroscopy

Example 3: 4-Hydroxy-5-iodo-phenyl-ethylcarbonyl-Asp-Tyr (SO3Na) -Met-Gly-Trp-Met-at-Asp-PheNH., 10 mg (22 μΜοί) N (4-hydroxy-5 -iodo-phenyl-ethylcarbonyloxy) -norborn-5-ene-2,3-dicarboximide are added to 10 mg (8.58 μΜοί) of Asp-Tyr (SO ₃ Na) -Met-Gly-Trp-Met-Asp-Phe- NH ₂ in 100 μΙ DMF and 100 μΙ pyridine in the presence of 2 μΙ Hünig base (N ₁ N'-diisopropylethylamine) and stirred for 8 hours at room temperature, the mixture injected into the HPLC, the appropriate fraction separated and lyophilized twice

 Yield: 9 mg 77.5%

Molar mass: 1 415

Amino Acid Analysis: Asp 2.0 (2.0), Tug 0.8 (1)

Met 1,95 (2), Gliy 1 (1), Trp 0,85 (1), Phe 0,94 (1)

The presence of the 4-hydroxy-5-iodo-phenyl-ethylcarbonyl residue could be demonstrated by mass spectroscopy: Sequence: C ₉ H ₇ O ₂ J 273.9495

C ₈ H ₇ OJ 245.9742

Example 4: 4-hydroxy-phenyl-ethylcarbonyl-Asp-Tyr (S0 ₃ Na) -Nle-Gly-Trp-Nle-Asp-Phe-NH2 20 mg (17.7 μΜοί) H-Asp-Tyr (SO ₃ Na ) N-Gly-Trp-Nle-Asp-Phe-NH ₂ are dissolved in 200 μΙ pyridine and 100 μΙ DMF, mixed with 6 μΙ (35 μΜ) Hünig base and treated with 11.8 mg (36 μΜοΙ) N ( p-hydroxy-phenyl-ethylcarbonyloxy) -norborn-5-ene-2,3-dicarboximide.

After eight hours of reaction, the batch is injected into the HPCL, the appropriate fraction separated, then concentrated, taken up with a little water and lyophilized.

Yield: 17 mg (69%) after twice lyophilization.

Amino acid analysis:

Asp 1,8 (2) Туг 0,8 (1) Never 2,2 (2) Gly 0,85 (1) Trp 0,75 (1) Phe 0,94 (1)

The presence of the p-hydroxy-phenylpropionyl residue was detected by mass spectroscopy (see Example 1).

HPLC conditions:

The substances are purified via Vydac RP 18 (15-20 m) in a column (20 mm i.d. χ 250 mm) in the following eluents:

Example 1 and 2:

Flow: 20 ml / min, UV detection: 220 nm

Eluent:

Example 1: A = 2,000 ml of 0,025 M ammonium acetate pH 6

B = methanol

Gradient: 25% B for 8 min to 26% retention time: 14.5-20.5 min collected

- Example 2: A: B, 50:50 retention time: 8.3-15.6 min collected Analysis: eluent see Examples 1 and 2

Example 3: R ₁ = 8.2 minutes uniform

- Example 4: R ₁ = 16.6 min uniform column dimension: 4.6 mm i. D. χ 250 mm, Vydac RP 18 (15-20 m)

Claims (1)

1. A process for the preparation of iodinated peptides and polypeptides, characterized in that free amino groups contained peptides or polypeptides of the general formula II or IM, in which AS ₁ to As _n any amino acid residues and As _x an amino acid with an H ₂ N group in the side chain and A and B in peptide chemistry conventional protecting groups or other organic radicals, with N ^ -hydroxy-S-iodo-phenyl-ethylcarbonyloxyJ-norborn-S-en ^ S-dicarboximide of the formula I, in the * for iodine ^125th stands to be implemented. CH ₂ -CH ₂ -CON