CZ280679B6 - Method of peptide radiolabelling - Google Patents

Abstract

The method of isotopically labeling peptides to obtain peptides containing isotopes of hydrogen is that the peptide with the free N-terminal amino group is heated in neutral or mildly alkaline medium at a pH of 7 to 11 at a temperature of 30 to 130. isotopes of hydrogen, wherein the isotopes of hydrogen are introduced to the carbon of the first, first and second, optionally first, second and third amine acids in the peptide chain. An advantage is a peptide which contains glycine in the 1-position or in the 1-position and 2-position

Description

Method of isotopic labeling of peptides

Technical field

The invention relates to a method for isotopically labeling peptides for obtaining peptides containing isotopes of hydrogen.

BACKGROUND OF THE INVENTION

Isotopically labeled organic compounds are a very important tool used in the research of their chemical transformations. In the case of biologically active peptides, isotopic labeling is virtually the only option used to study the mechanism of their action and to monitor their fate in a living organism. Hitherto known methods of introducing an isotopic label into peptides include a) non-specific exchange of hydrogen atoms - the so-called Wilzbach labeling, b) addition of hydrogen to unsaturated bonds, c) catalytic exchange of halogen atoms on the aromatic nucleus. Only by these methods can a labeled substance that is not chemically different from a non-labeled substance can be obtained relatively easily. Other ways of labeling either alter the nature of the study substance - by introducing radioactive atoms initially in the molecule (eg iodine isotopes) or by attaching blocks containing a radiolabel - or are synthetically very demanding - synthesis is based on precursors (usually amino acids) already containing radioactive isotopes ( ³ H, ¹ H, ¹³ C).

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides a method for isotopically labeling peptides for obtaining peptides containing isotopes of hydrogen, which comprises heating a peptide having a free N-terminal amino group in a neutral or slightly alkaline environment at pH 7-11 at 30-130 ° C in the presence of water. with an altered representation of hydrogen isotopes, wherein the isotopes of hydrogen are introduced at the α-carbon of the first, first and second, and optionally the first, second and third amino acids in the peptide chain. Preferably, the peptide comprises glycine at position 1 or at positions 1 and 2. The peptide thus obtained can be used as one of the fragments in peptide synthesis. The method of labeling peptides according to the invention is based on the finding that peptides in aqueous medium under neutral or different alkaline conditions (pH 7-11) and elevated temperature (30-130 ° C) are subject to racemization of amminothermal amino acids. or three amino acids. Thus, any peptide with a free α-amino group can be heated in a neutral to slightly alkaline solution in water with an altered representation of hydrogen isotopes to yield a mixture of distereoisomeric peptides in which the hydrogen is on the α-carbon of the first amino acid ( second and third amino acids) exchanged for an isotope or mixture of hydrogen isotopes contained in the water used.

The diastereoisomeric peptides obtained when the terminal amino acid is not glycine can be separated by high pressure liquid chromatography (HPLC) to obtain a peptide with the same configuration of all asymmetric centers as the starting material very easily. This method is particularly preferred for a peptide comprising

-1C 280679 B6 at position 1 glycine (optionally containing glycine at positions 1, 2 or 1, 2, 3), since there is no mixture of diastereoisomers in this case (and therefore no HPLC purification is required) and the labeling yield is higher as exchange two protons in the first amino acid. The use of amino-terminal glycine can also be recommended for labeling substances that contain glycine within their amino acid sequence. In these cases, the synthesis of both the amino-terminal glycine (I) containing peptide and the amino-terminal (II) peptide must be synthesized. Fragment I is then subjected to the introduction of hydrogen isotopes as described above, coupled thereto using known peptide synthesis methods, fragment II, and, after deprotection, the specifically labeled desired peptide is obtained. The advantage of the marking method according to the invention is a very simple embodiment, low cost of the required chemicals.

The following examples illustrate but do not limit the process of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Explanation of abbreviations used in the examples:

TLC = thin-layer chromatography,

HPLC = high pressure liquid chromatography.

Example 1

Η- ^(a-2H) -Phe-Met-Arg-Phe-OH

Tetrapeptide hydrochloride H-Phe-Met-Arg-Phe-OH (30 mg) was dissolved in D ₂ O (10 mL), the pH of the solution was adjusted to 7.3 with 1 mol / L potassium hydroxide solution and the solution was sealed. The solution was applied to a Silasorb Cg column (25x2.4 cm) and gradient elution (30-50% acetonitrile in 0.1% trifluoroacetic acid over 50 min) of the obtained fraction. were heated under vacuum and lyophilized. 8 mg of a substance was obtained whose retention time on a Separon SIC ₁₈ column (25x0.4 cm) was identical to the retention time of the starting material. ¹ H NMR analysis showed that this material contained only 35% of ¹ H hydrogen on the α-carbon of the aminoterminal phenylalanine. The obtained peptide was pure by TLC (thin layer chromatography) [R _F 0.24 (2-butanol - 98% formic acid - water, 75:: 13.5: 11.5 (system A)], 0.01 [2 -butanol - 25% aqueous ammonia

- water, 85: 7.5: 7.5 (system B)], 0.09 [1-butanol-acetic acid - water, 4: 1: 1 (system C) J, 0.58 [1-butanol - pyridine

- acetic acid - water, 15: 10: 3: 6 (System D)], electrophoretic analysis ^(His E 5.7 = 0.44, E ⁼ 2R4 ^Hls °> ^{82) and} HPLC (see above) and coincide with the all of these starting material tests. Amino Acid Analysis: Phe 1.96 (2), Met 0.88 (1), Arg 1.04 (1). FAB MS: 601.3 + 600.3 (M + H < + & gt ^; ).

Example 2

Η- (α- ² H ₂ ) Gly-Gly-Phe-Met-OH

The free tetrapeptide H-Gly-Gly-Phe-Met-OH (5 mg) was dissolved in D ₂ O (5 mL), the pH of the solution was adjusted to 9.25 with 1 M potassium hydroxide solution, and the solution was sealed. The vial was heated to 120 ° C for 23 h. After this time, the solution was loaded onto a Silasorb C ₈ column (25 x 2.4 cm) and the fractions obtained by gradient elution were heated in vacuo and lyophilized. 5 mg of substance was obtained whose retention time on a Separon SIC ₁₈ column (25 x 0.4 cm) was the same as that of the starting material. 1 H NMR analysis showed that this material contained only 20% hydrogen on the α-carbon of the aminoterminal glycine. The peptide obtained was pure by TLC [Rp 0.28 (A), 0.03 (B), 0.32 (C), 0.55 (D)], electrophoretic analysis (E ^His 5.7 = 0/01 / W ^G1 ^ 24 =

0.96) and HPLC (see above) and was consistent with the starting material in all of these assays. Amino Acid Analysis: Phe 0.96 (1), Gly 2.03 (2), Met 0.89 (1). FAB MS: 413.2 (M + H < + & gt ^; ).

Example 3

E- (α- ² H2) Gly- (α- ² H2) Gly- (α- ² H) Phe-Met-OH

The free tetrapeptide H-Gly-Gly-Phe-Met-OH (20 mg) was dissolved in D ₂ O (5 mL), the pH of the solution was adjusted to 11.9, and the solution was heated to 120 ° C in a sealed vial. After this time, the solution was loaded onto a Silasorb C ₈ column (25 x 2.4 cm) and gradient elution (0-40% methanol in 0.1% trifluoroacetic acid over 5 min and 40-50% methanol in 0, 1% trifluoroacetic acid over 50 min) The fractions obtained were heated under vacuum and lyophilized. 9 mg of substance was obtained, whose retention time on a Separon SIC ₁₈ column (25 x 0.4 cm) was identical to the retention time of the starting material. 1 H NMR spectroscopy showed that this material contained only traces of ¹ H hydrogen at the α-carbon of the amino-terminal glycine and α-carbon glycine at the 2-position and 10% hydrogen · α · Η at the α-carbon of phenylalanine at the 3-position. The obtained peptide was pure by TLC [Rp 0.28 (A), 0.03 (B), 0.32 (C), 0.55 (D)], electrophoretic analysis (E ^H - * - ^s 57 = 0) ^.Alpha.1 , ^{.delta. (G} @ 23 = 0.96) and HPLC (see above) and coincided with the starting material in all these tests. Amino Acid Analysis: Phe 1.03 (1), Gly 2.07 (2), Met 0.88 (1), FAB MS: 416.2 (M + H ⁺ ).

Example 4

H-Tyr- (α- ² H2) Gly- (α- ² H2) Gly- (α- ² H) Phe-Met-OH

The peptide prepared in Example 3 (5 mg) was dissolved in water,

The pH of the solution was adjusted to 9.5 and Boc-Tyr-ONSu solution (10 mg) was added to the obtained solution. The mixture was stirred at room temperature until the disappearance of the starting peptide (TLC) analysis). After the solution was evaporated, the residue was dissolved in a mixture of trifluoroacetic acid and anisole (95: 5) and after 20 min the solution was evaporated, the residue was dissolved in 3 mol / L acetic acid and loaded onto a Vydac C ₁₈ column (25 χ 1 cm). Gradient elution (20-50% acetonitrile in 0.05% trifluoroacetic acid over 40 min) obtained fractions were heated under vacuum and lyophilized. 4.7 mg of a substance was obtained whose retention time on a Separon SIC ₁₈ column (25 x 0.4 cm) was the same as that of the starting material. NMR analysis showed that it had the same hydrogen content on the α-carbons of glycine at position 2 and 3 and phenylalanine at position 4 as the starting tetrapeptide. The peptide was pure by TLC [R _p 0.32 (A) 0.10 (B) 0.41 (C) 0.57 (D) J electrophoretic analyzes (EHIS ^ _ Q i _of Q _R E ^G1 (24 = 0/67) and HPLC (see above) and met all methionine enkephalin standard in all these tests. Amino Acid Analysis: Phe 1.03 (1), Gly 2.10 (2), Met 0.87 (1), Tyr 0.93 (1). FAB MS: 579.2 (M + N < + & gt ^; ).

Industrial applicability

The method of the invention can be used in basic research to study the metabolism and distribution of biologically active peptides in a living organism.

Claims (2)

PATENT CLAIMS A method of isotopically labeling peptides for obtaining peptides containing isotopes of hydrogen, characterized in that the peptide with a free N-terminal amino group is heated in a neutral or slightly alkaline medium at pH 7-11 at 30 to 130 ° C and in the presence of water with altered proportions of hydrogen isotopes, wherein the isotopes of hydrogen are introduced at the α-carbon of the first, first and second, respectively the first, second and third amino acids in the peptide chain. The method of claim 1, wherein the labeled peptide is a peptide containing glycine at position 1 or at positions 1 and 2.