WO1992007265A1

WO1992007265A1 - Selective peptide labeling with hydrogen isotopes

Info

Publication number: WO1992007265A1
Application number: PCT/US1991/007658
Authority: WO
Inventors: Michal Lebl; Zhanna D. Bespalova; Nikolai F. Sepetov
Original assignee: Michal Lebl; Bespalova Zhanna D; Sepetov Nikolai F
Priority date: 1990-10-18
Filing date: 1991-10-18
Publication date: 1992-04-30
Also published as: CZ507690A3; AU8953691A; CZ280679B6

Abstract

A simple method of hydrogen isotope introduction into the α-position of N-terminal amino acid in the peptide chain comprising of heating the peptide in solution in the presence of water containing the hydrogen under neutral or slightly basic conditions was found. Increase of the solution pH leads to the introduction of hydrogen isotope into up to three N-terminal amino acids. This N-terminal-modified peptide can be used in further synthetic reactions. The method was demonstrated in the synthesis of pentadeuterated methionine enkephalin.

Description

SELECTIVE PEPTIDE LABELING WITH HYDROGEN ISOTOPES |

1. FIELD OF THE INVENTION The present invention is related to methods c introducing hydrogen isotopes in a peptide.

Introduction of such hydrogen isotopes can provide detectable labels.

2. BACKGROUND OF THE INVENTION Isotopically labeled organic compounds are very important tools used in the investigation of their chemical transformations. In the case of biologically active peptides, isotopic labeling is basically the only method used in mechanism studies and in following their fate in the living organism. The known ways of introduction of the isotopic label comprise: (i) non-specific exchange of hydrogen atoms — the so called ilzbach method; (ii) addition of hydrogens to unsaturated bonds; (iii) catalytic exchange of halogen atoms on the aromatic nucleus;

(iv) total synthesis of the organic molecule starting from a labeled precursor. These methods can produce compounds chemically indistinguishable from the native unlabeled species. Other ways of labeling — for example, introduction of atoms originally not included in the molecule (such as iodine atoms) , or adjoining the moieties containing the label — change the character of the original molecule and results obtained with these modified compounds might be misleading.

It is an object of the present invention to overcome the deficiencies of the known methods of introducing an isotope of hydrogen into a peptide. 3. SUMMARY OF THE INVENTION A method for introducing an isotope of hydrogen into a peptide comprises heating the peptide in solution of pH not less than 7, in the presence of water containing the hydrogen isotope. The hydrogen isotope is present in the water in a ratio desired for the ratio of isotopic hydrogen to be introduced into the peptide.

4. DETAILED DESCRIPTION OF THE INVENTION

The method of introducing an isotope of hydrogen according to the invention is based on the finding that peptides in aqueous solutions under neutral or slightly basic conditions and increased temperature are racemized at the α-carbon of amino- ter inal amino acids. As used herein, the term peptide is intended to include oligopeptides, polypeptides, proteins and proteinaceous compounds. A free N-terminal amino group potentiates introduction of hydrogen at the α-carbon position. Incorporation of hydrogen results in randomization of configuration at the α-carbon.

To introduce a hydrogen isotope into a peptide, the peptide in aqueous solution is heated for as long as is necessary to introduce the hydrogen isotope. Although any solvent in which both the peptide and water are soluble or miscible may be used, water is preferred. More preferably, if possible, the water is quantitatively (i.e., about 100%) composed of the hydrogen isotope desired for introduction on the peptide. The extent of isotope incorporation of the α-carbon will be proportional to the ratio of hydrogen isotopes in the water. According to the invention, a temperature range of about 30-l20°C can be used; in a specific embodiment, the peptide is heated to about 120°C. Preferably the solution is heated in a closed reaction vessel to prevent evaporation. In a specific embodiment, a sealed ampoule is used. In another embodiment, a teflon-stoppered tube (Pierce) is used. Introduction of an inert gas such as nitrogen can prevent oxidative degradation during heating, although this is not essential. In specific embodiments, the solution is heated for about 20-50 hours. A suitable pH range for the solution is about pH 7-12. The amount of hydrogen isotope incorporated in the peptide increases with higher pH, i.e., more basic conditions. Selection of proper conditions allows labeling of one, two or three amino acids. As used herein, the first amino acid, at position 1, is the most N-terminal amino acid. The second amino acid at position 2 is next to the first amino acid, toward the C-terminus. The third amino acid at position 3 is next to the second amino acid, towards the C-terminus.

The hydrogen isotope, which may be ^JH (hydrogen) , ²H (deuterium, D) , or ³H (tritium, T) is donated by water molecules present in the solution. The water molecules are composed of the hydrogen isotope and oxygen, e.g., H,0, D₂0, or HTO (although T,0 can theoretically be used, because this molecule is unstable, as a practiced matter both H and T will be present in the water) . Preferably the isotope of hydrogen introduced into the peptide is deuterium or tritium, since these isotopes are readily detected by proton NMR (deuterium) , scintillation counting (tritium) , autoradiography (tritium) and mass spectroscopy (deuterium and tritium) . Uses for peptides in which deuterium or tritium have been incorporated are well known in the art. In other instances, however, it may be desireable to introduce hydrogen. It is thus possible to heat any peptide containing a free N-terminal amino group in neutral to slightly basic aqueous solution with altered ratio of hydrogen isotopes and obtain in this way the mixture of diastercoisomeric peptides, in which the hydrogen on α-carbon of the first amino acid (and in the case of more drastic reaction conditions also on the α- carbon of the second and third amino acids) is substituted by the isotope or mixture of isotopes of hydrogen which are contained in the water. The extent of substitution is proportional to the ratio of the hydrogen isotopes in the water. Diastereoisomeric peptides can be separated, e.g., by HPLC, and a peptide with the same configuration of all asymmetrical centers as the starting peptide can be obtained in this way very easily. A particular advantage of the invention is that hydrogen introduced at the α-carbon position is not labile under normal conditions, e.g., physiological conditions. Thus the hydrogen isotope label remains stable.

It will be recognized that in the present invention water acts as a hydrogen donor to the α- carbon under conditions of elevated temperature and neutral or basic pH. Other compounds known in the art or discovered that can donate hydrogen to the α-carbon of an N-terminal amino acid under those conditions can also be used in the practice of the invention, and are contemplated to fall within the scope of the invention.

The present method of introducing hydrogen at the α-carbon is especially advantageous in the cases of peptides containing glycine in position 1 (or those containing glycine in positions 1 and 2, or 1, 2, and 3), since these peptides do not give rise to diastereoisomeric mixtures and therefore purification is not needed. At the same time the labeling yield is higher because it is possible to exchange two protons in the glycine moiety.

The method of the invention can be used to incorporate a labeled amino acid within a peptide sequence. Preferably the labeled amino acid is glycine, so no separation of diastereoisomers is necessary. In these cases both a carboxy-terminal fragment containing amino-terminal glycine (I) and an amino-terminal fragment (II) are synthesized. The present method may be then used to introduce a hydrogen isotope into fragment I. Fragment II can then be connected to the free N-terminous of fragment I by established methods of peptide synthesis. Removal of side chain protecting groups completes the peptide preparation.

The notable advantages of the present method for introducing an isotope of hydrogen in a peptide especially for selective peptide labeling are simplicity and economy. The following examples are provided as illustration of the methods of the invention and are not intended as limitations.

5. EXAMPLES 5.1. EXAMPLE 1; H-fα-²H)-Phe-Met-Arσ-Phe-OH The tetrapeptide hydrochloride H-Phe-Met- Arg-Phe-OH (30 mg) was dissolved in D₂0 (10 ml) , the pH of solution was adjusted to 7.3 by addition of 1 M KOH, and the solution was heated to 120°C for 47 h in a sealed ampoule. After this treatment the solution was introduced onto a column of Silasorb C₈ (25 x 2.4 cm) . The reaction products were separated by reverse phase chromatography with gradient elution (30 - 50% of acetonitrile in 0.1% trifluoroacetic acid in 50 min) . Fractions were lyophilized. A peptide with the same retention time (on Separon SlC_jg, 25 x 0.4 cm) as the starting material was obtained (8 mg) . Proton NMR spectroscopy showed that this compound contains only 35% of hydrogen *H on α-carbon of amino terminal phenylalanine. Obtained peptide was pure according to TLC (R_F 0.24 (SI), 0.01 (S2), 0.09 (S3), 0.58 (S4)), electrophoresis (E"~ ₅.₇ 0.44, E^GIy ₂.₄ 0.82) and HPLC (see above) , and was identical in all these tests with the starting compound. Amino acid analysis: Phe 1.96 (2), Met 0.88 (1), Arg 1.04 (1). FAB MS: 601.3 and 600.3 (13:7) (M+H); calculated for peptide containing *H isotopes: 600.3.

5.2. EXAMPLE 2: H-(α-²H- Gly-Glv-Phe-Met-OH The tetrapeptide H-Gly-Gly-Phe-Met-OH (5 mg) was dissolved in D₂0 (5 ml) , the pH of solution was adjusted to 9.25 by addition of 1 M KOH, and the solution was heated to 120°C for 23 h in a sealed ampoule. The solution was worked up by reverse phase chromatography as described above (the gradient was 0- 40% methanol in 0.1% trifluoroacetic acid in 5 min, followed by 40 - 50% methanol in 0.1% TFA in 50 min). The product (5 mg) contained only 20% of hydrogen Η on the N-terminal glycine α-carbon according to proton NMR. TLC (R_HJ, 0.28 (SI), O.03 (S2) , 0.32 (S3), 0.55 (S4)), electrophoresis (E^ffis ₅.₇ 0.01, E^Gly ₂₄ 0.96) and HPLC showed identity with the starting molecule. Amino acid analysis: Phe 0.96 (1), Gly 2.03 (2), Met 0.89 (1). FAB MS: 413.2 (M+H⁺) ; calculated for peptide containing ^lE isotopes: 411.2.

5.3. EXAMPLE 3: H-fα-²H,1Glv-fα-²-H,lGlv- ( ct-²H)Phe-Met-OH

The tetrapeptide H-Gly-Gly-Phe-Met-OH (20 mg) was treated as described in Section 5.2.. above, but the pH of the D₂0 (5 ml) solution was adjusted to 11.9 (120°C, 24 h) . The same work-up procedure afforded 9 mg of compound, identical to the starting peptide (TLC, electrophoresis, HPLC) . Proton NMR spectroscopy showed that the product contained only traces of hydrogen 'H on α-carbons of amino terminal glycine and glycine in position 2, and 10% of hydrogen ^]H on α-carbon of phenylalanine in position 3. Amino acid analysis: Phe 1.03 (1), Gly 2.07 (2), Met 0.88 (1). FAB MS: 416.2 (M+H⁺) ; calculated for peptide containing all *H isotopes: 411.2.

5.4 EXAMPLE 4: H-Tyr-(α-²H₂)Gly-(α-²H₂)Gly- fα-²H^Phe-Met-OH

Peptide prepared as described above in Section 5.3. (5 mg) was dissolved in water, the pH of the solution was adjusted to 9.5 and a solution of Boc-Tyr-ONSu (10 mg) was added. The mixture was stirred at room temperature until the starting peptide disappeared (TLC analysis) . The solution was evaporated, and the residue was dissolved in a mixture of trifluoroacetic acid and anisole (95:5). After 20 minutes the mixture was evaporated. The residue was dissolved in 3 M acetic acid and introduced onto a column of Vydac C₁₈ (25 x l cm) . Reaction products were separated by gradient elution (20 - 50% of acetonitrile in 0.05% trifluoroacetic acid in 40 min) . Fractions were lyophilized. The labeled pentapeptide (4.7 mg) was identical to the standard Met-enkephalin by HPLC analysis on Separon SIC₁₈, 25 x 0.4 cm. Proton NMR spectroscopy confirmed the same hydrogen isotope content as found in the tetrapeptide. The pentapeptide was pure according to TLC (R 0.32 (SI) 0.10 (S2) , 0.41 (S3), 0.57 (S4) ) , electrophoresis (E^ffis ₅.₇ 0.01, E^Gly ₂.₄ 0.67) and HPLC. Amino acid analysis: Phe 1.03 (1), Gly 2.10 (2), Met 0.87 (1), Tyr 0.93 (1). FAB MS: 579.2 (M+H⁺) ; calculated for peptide containing all ^JH isotopes: 574.2.

5.5 DISCUSSION The foregoing examples clearly demonstrate that an isotope of hydrogen, e.g. deuterium, can be introduced at the N-terminal α-carbon position of a peptide. In each case a chemically identical product — as determined by analytical HPLC, electrophoresis and TLC — was obtained, but proton NMR spectroscopy and fast ion bombardment mass spectroscopy (FAB MS) unequivocally demonstrated the presence of deuterium. Deuterium is invisible in proton NMR, thus its presence results in decrease or disapperance of signal. Deuterium has an atomic weight for 2; H hydrogen has atomic weigh of 1, thus each substitution of deuterium for hydrogen results in an increase of 1 in molecular weight. As shown in the examples, the increase in molecular weight of the product exactly correlated with the number of deuterium atoms introduced. As shown in Example 4, the hydrogen isotope can be incorporated in the interior of a peptide sequence by adding additional amino acids or peptide sequences to the free N-terminus after the peptide has been labeled. In this way, deuterated Met-enkephalin was prepared.

5.6 GENERAL METHODS Thin layer chromatography was carried out on silica gel coated plates (Silufol, Kavalier, Czechoslovakia) in the following systems: 2-butanol- 98% formic acid water (10:3:8) (SI); 2-butanol-25% ammonia-water (85:7.5:7.5) (S2) ; 1-butanol-acetic acid-water (4:1:1) (S3); and 1-butanol-acetic acid- pyridine-water (15:3:10:6) (S4) . Paper electrophoresis was performed in a moist chamber in 1 M acetic acid (pH 2.4) and in pyridine-acetate buffer (pH 5.7) on Whatman 3MM paper at 20 V/cm for 60 minutes. Spots in TLC and electrophoresis were detected with ninhydrin or by the chlorination method. Samples for amino acid analysis were hydrolyzed with 6 M HC1 at 105°C for 20 h and analyzed on an amino acid analyzer (T 339 Mikrotechna Praha, Czechoslovakia or D-500, Durrum Corp., U.S.A.). Fast atom bombardment mass spectra (FAB MS) were obtained on a ZAB-EQ spectrometer (VG Analytical Ltd. , Manchester) with xenon at 8 kV as the bombarding gas. High performance liquid chromatography (HPLC) was carried out on an SP- 8800 instrument equipped with an SP-8450 detector and SP-4290 integrator (all from Spectra Physics, Santa Clara, USA) or on an HP 1090 (Hewlett Packard) . Preparative liquid chromatography was carried out on the above described equipment of Spectra Physics using a Vydac 218TP510 column (5 μm, 250 x 10 mm, The Separations Group, Hesperia, CA) , or Silasorb C₈ column (10 μm, 250 x 24 mm, Lachema, Brno, Czechoslovakia) . Analytical HPLC was performed on a Separon SIC₁₈ column (5 μm, 250 x 4 mm, Tessek, Prague, Czechoslovakia) . 'H NMR spectra were obtained on Bruker WH-500 (500 MHz) .

Claims

What is claimed is:

1. A method for introducing an isotope of hydrogen into a peptide comprising heating the peptide in solution of pH not less than 7, in the presence of water composed of the isotope of hydrogen.

2. The method according to claim 1 wherein the pH of the solution is pH 7-12.

3. The method according to claim 1 wherein the solution is heated to about 30-120°C.

4. The method according to claim 3 wherein the solution to about 120°C.

5. The method according to claim 1 wherein the solution is heated for about 20 to 50 hours.

6. The method according to claim 1 wherein the isotopic hydrogen is deuterium or tritium.

7. The method according to claim 1 wherein the peptide contains an N-terminal glycine.

8. The method according to claim 7 wherein the peptide is selected from the group consisting of: (i) a peptide with glycine at position 1; (ii) a peptide with glycines at positions 1 and 2; and (iϋ) a peptide with glycines at positions

1, 2 and 3.

9. The method of claim 1 wherein the peptide into which hydrogen was introduced comprises a racemic mixture of peptides.

10. The method of claim 1 further comprising separating racemic products.

11. A method for introducing an isotope of hydrogen into a peptide comprising heating the peptide to 30°-120°C in solution of about pH 7-12 in the presence of water composed of the isotope of hydrogen.

12. The method according to claim 11 wherein the solution is heated for about 20 to 50 hours.

13. The method according to claim 11 wherein the isotope of hydrogen is deuterium or tritium.

14. The method of claim 11 wherein the peptide into which hydrogen was introduced comprises a racemic mixture of peptides.

15. The method according to claim 14 further comprising separating racemic products.

16. A method for introducing an isotope of hydrogen into a peptide containing an N-terminal glycine comprising heating the peptide to 30°-120°C in solution of pH 7-12 in the presence of water composed of the isotope of hydrogen.