Process for the specific isotopic labeling of methyl groups of Val, Leu and Ile

Abstract

A process for the specific isotopic labeling of an amino acid selected from Valine (Val), Leucine (Leu), and Isoleucine (Ile), in proteins and biomolecular assemblies by introducing, in a medium containing bacteria overexpressing a protein, an acetolactate derivative of Formula I of the application.

Claims

1. A process for the specific isotopic labeling of an amino acid selected from Valine (Val), Leucine (Leu), and Isoleucine (Ile), in proteins and biomolecular assemblies comprising: a) introducing, in a medium containing bacteria overexpressing a protein, a composition consisting essentially of an acetolactate derivative of Formula I: ##STR00034## wherein: X is .sup.1H or .sup.2H (D), each Y is independently from the others .sup.12C or .sup.13C, R.sup.1 is a methyl group in which the carbon atom is .sup.13C or .sup.12C and the hydrogen atoms are independently from each other .sup.1H or .sup.2H (D), R.sup.2 is either a methyl group in which the carbon atom is .sup.13C or .sup.12C and the hydrogen atoms are independently from each other .sup.1H or .sup.2H (D), or an ethyl group in which the carbon atoms are independently from each other .sup.13C or .sup.12C and the hydrogen atoms are independently from each other .sup.1H or .sup.2H (D), with the proviso that: the hydrogen atoms of R.sup.1 and the hydrogen atoms of R.sup.2 are not all, at the same time, either .sup.1H or .sup.2H (D).

2. The process of claim 1 further comprising: b) overexpression of the protein by the bacteria contained in the medium, and c) purification of the protein.

3. The process of claim 1, wherein in the acetolactate derivative of Formula I: R.sup.1 is chosen from the following groups: .sup.12CH.sub.3, .sup.12CD.sub.3, .sup.13CH.sub.3, .sup.13CD.sub.3, .sup.13CHD.sub.2, .sup.13CH.sub.2D, and R.sup.2 is chosen from the following groups: .sup.12CH.sub.3, .sup.12CD.sub.3, .sup.13CH.sub.3, .sup.13CD.sub.3, .sup.13CHD.sub.2, .sup.13CH.sub.2D, .sup.12CH.sub.3.sup.12CD.sub.2, .sup.12CD.sub.3.sup.12CD.sub.2, .sup.13CH.sub.3.sup.12CD.sub.2, .sup.13CH.sub.3.sup.13CD.sub.2, .sup.13CD.sub.3.sup.13CD.sub.2, .sup.13CHD.sub.2.sup.13CD.sub.2, .sup.13CH.sub.2D.sup.13CD.sub.2, .sup.13CHD.sub.2.sup.12CD.sub.2, .sup.13CH.sub.2D.sup.12CD.sub.2.

4. The process of claim 1, wherein the acetolactate derivative of Formula I is selected from the following compounds: 2-hydroxy-2-(.sup.13C)methyl-3-oxo-4(.sup.2H.sub.3)butanoic acid, 2-hydroxy-2-(.sup.2H.sub.3)methyl-3-oxo-4(.sup.13C)butanoic acid, 2-(.sup.2H.sub.5)ethyl-2-hydroxy-3-oxo-4-(.sup.13C)methylbutanoic acid, 1,2,3,4-(.sup.13C)-2-(.sup.2H.sub.5)ethyl-2-hydroxy-3-oxobutanoic acid, 1,2,3-(.sup.13C)-2-(.sup.13C)methyl-2-hydroxy-3-oxo-4-(.sup.2H.sub.3)butanoic acid, 1,2,3,4-(.sup.13C)-2-(.sup.2H.sub.3)methyl-2-hydroxy-3-oxobutanoic acid, 1,2,3-(.sup.13C)-2-(1-(.sup.2H.sub.2), .sup.13C.sub.2)ethyl)-2-hydroxy-3-oxo-4-(.sup.2H.sub.3)butanoic acid, 3,4-(.sup.13C)-2-(.sup.13C)methyl-2-hydroxy-3-oxo-4-(.sup.2H.sub.3)butanoic acid, 3,4-(.sup.13C)-2-(.sup.2H.sub.3, .sup.13C)methyl-2-hydroxy-3-oxobutanoic acid, 3,4-(.sup.13C)-2-(1-(.sup.2H.sub.2), .sup.13C.sub.2)ethyl-2-hydroxy-3-oxo-4-(.sup.2H.sub.3)butanoic acid, 3,4-(.sup.13C)-2-(.sup.2H.sub.5, .sup.13C.sub.2)ethyl-2-hydroxy-3-oxobutanoic acid, and 3,4-(.sup.13C)-2-(1-(.sup.2H.sub.2),.sup.13C.sub.2)ethyl-2-hydroxy-3-oxobutanoic acid.

5. The process of claim 1, wherein the acetolactate derivative of Formula I is selected from the following compounds: 2-hydroxy-2-(.sup.2H.sub.2, .sup.13C)methyl-3-oxo-4-(.sup.2H.sub.3)butanoic acid, 2-hydroxy-2-(.sup.2H.sub.3)methyl-3-oxo-4-(.sup.2H.sub.2, .sup.13C)butanoic acid, 2-(.sup.2H.sub.5)ethyl-2-hydroxy-3-oxo-4-(.sup.2H.sub.2, .sup.13C)butanoic acid, and 2-(1-(.sup.2H.sub.2),2-(.sup.2H),2-(.sup.13C))ethyl-2-hydroxy-3-oxo-4-(.sup.2H.sub.3)butanoic acid.

6. The process of claim 1, wherein the acetolactate derivative of Formula I is selected from the following compounds: 2-hydroxy-2-(.sup.2H, .sup.13C)methyl-3-oxo-4-(.sup.2H.sub.3)butanoic acid, 2-hydroxy-2-(.sup.2H.sub.3)methyl-3-oxo-4-(.sup.2H, .sup.13C)butanoic acid, and 2-(.sup.2H.sub.5)ethyl-2-hydroxy-3-oxo-4-(.sup.2H, .sup.13C)butanoic acid.

7. The process of claim 1, wherein said process specifically isotopically labels Valine in proteins and biomolecular assemblies.

8. The process of claim 1, wherein said process specifically isotopically labels Leucine in proteins and biomolecular assemblies.

9. The process of claim 1, wherein said process specifically isotopically labels Isoleucine in proteins and biomolecular assemblies.

10. The process of claim 1, wherein said process specifically isotopically labels methyl groups of Leucine and Valine in proteins and biomolecular assemblies.

11. The process of claim 1, wherein said process specifically isotopically labels 2-methyl groups of Isoleucine in proteins and biomolecular assemblies.

12. A process for analyzing a protein by NMR, comprising labeling a protein to be analyzed by the process of claim 1.

Description

(1) In order that the invention be better understood, an example of carrying out the labeling process of the invention is given below. This example is only illustrative, and in no way limitative of the invention.

1. Synthesis of Selectively Methyl-Labeled Acetolactate

Ethyl 2-(13C)methyl-3-oxobutanoate

(2) A mixture of 12.15 mL (95.4 mmol) of ethyl 3-oxobutanoate (A), 14.5 g (104.9 mmol) K.sub.2CO.sub.3 and 15.0 g (104.9 mmol) .sup.13C-methyl iodide (Cambridge Isotope Laboratories, Inc.) in 120 mL of absolute ethanol was heated at 40 C. under argon for 90 h. After filtration, the filtrate was concentrated in vacuo to afford 12.30 g (yield 89%) of product, which was sufficiently pure to be used without further purification. .sup.1H NMR (CDCl.sub.3); 4.21 (q, J=7.1 Hz, 2H), 3.51 (dq, J=7.2, 4.4 Hz, 1H), 2.25 (s, 3H), 1.35 (dd, J=130.4, 7.2 Hz, 3H), 1.29 (t, J=7.1 Hz, 3H).

Ethyl 2-hydroxy-2-(13C)methyl-3-oxobutanoate (B)

(3) Hydroxylation reaction was carried out by freshly prepared dimethyldioxirane in presence of Nickel(II) ions. To a mixture of 50 mg (0.345 mmol) of ethyl 2-(.sup.13C)methyl-3-oxobutanoate in 3 mL of distilled water, was added successively 8.6 mg (0.035 mmole) of Ni(OAc).sub.2.4H.sub.2O and, at 0 C., 20 mL of an untitrated solution of dimethyldioxirane (0.05-0.10 M) in acetone. The resulting solution was allowed to warm to room temperature and stirred for 24 hours. The organic solvant was then evaporated in vacuo and the resulting aqueous residue was extracted with dichloromethane (four times). The organic extract was dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford 51 mg (0.317 mmol; 92% yield; >90% conversion) of ethyl 2-hydroxy-2-(.sup.13C)methyl-3-oxobutanoate as a colorless liquid which was pure enough to be used without further purification.

(4) NMR spectroscopy: .sup.1H NMR (CDCl.sub.3); 4.25 (q, J=7.1 Hz, 2H), 2.27 (s, 3H), 1.58 (d, .sup.1J.sub.H-13C=130.3 Hz, 3H), 1.29 (t, J=7.1 Hz, 3H).

2-hydroxy-2-(13C)methyl-3-oxo-4-(2H3)-butanoate (or 2-(13C)methyl-4-(2H3)-acetolaetate) (Compound of Formula 4)

(5) Deprotection and exchange of the protons of the methyl group in position 4 of ethyl 2-hydroxy-2-(.sup.13C)methyl-3-oxobutanoate (B) were achieved in D.sub.2O at pH 13. Typically, 300 mg of B was added to 24 mL of a 0.1 M NaOD/D.sub.2O solution. The deprotection was immediate as observed by NMR spectroscopy. The completion of the exchange on the 4-methyl was also followed in real time by NMR spectrometry. 971% of protons of terminal methyl groups have been exchanged after 30 min while the methyl subsistent in position 2 remains protonated at a level of 981%. As the deprotection reaction consumes hydroxide ions, the pH and consequently the deuterium exchange rate decreases during the reaction. Once the exchange was complete, the solution was adjusted to neutral pH with DCl and 2 mL of 1 M TRIS pH 8 in D.sub.2O was added. The solution of the compound of formula 4 was then stored at 20 C. until required.

(6) Reaction Scheme of the Protocol for the Production of U[.sup.2H], U[.sup.15N], Leu/Val-[.sup.13C.sup.1H.sub.3].sup.proS Labeled Proteins.

(7) ##STR00032## ##STR00033##

(8) Detailed protocol for the chemical synthesis of 2-(.sup.13C)methyl-4-(.sup.2H.sub.3)-acetolactate is presented above. .sup.13C nuclei are displayed in italic bold. The stereochemistry, following the incorporation of .sup.13C.sup.1H.sub.3 group into acetolactate, in the different intermediates of Leu/Val biogenesis pathway is indicated on the figure (assuming growth in M9/D.sub.2O based culture medium). Each biosynthetic intermediate has been named according to the Kyoto Encyclopedia of Genes and Genomes. The enzymes responsible for catalyzing reaction are indicated by EC number. EC 1.1.1.86: ketol-acid reductoisomerase; EC 4.2.1.9: dihydroxy-acid dehydratase; EC 2.6.1.42: branched-chain amino acid aminotransferase; EC 2.3.3.13: 2-isopropylmalate synthase; EC 4.2.1.33: 3-isopropylmalate dehydratase; EC 1.1.1.85: 3-isopropylmalate dehydrogenase. Further information on the Leu/Val metabolic pathway can be found online: http://www.genome.jp/kegg/.

2. Overexpression of Methyl Stereospecifically Labeled Proteins in E. coli

(9) Optimization of the Incorporation of Acetolactate in Overexpressed Protein.

(10) Initial experiments to determine the level of acetolactate incorporation into overexpressed proteins were performed using ubiquitin as a model system. E. coli BL21(DE3) cells were transformed with a pET41c plasmid carrying the human His-tagged ubiquitin (pET41c-His-Ubi) gene and transformants were grown in M9/D.sub.2O media containing 1 g/L .sup.15ND.sub.4Cl, and 2 g/L of U-[.sup.2H], U-[.sup.13C], glucose. When the optical density (O.D.) at 600 nm reached 0.8, a solution containing unlabeled acetolactate was added. After an additional 1 h, protein expression was induced by the addition of IPTG to a final concentration of 1 mM. Induction was performed for 3 hours at 37 C. Ubiquitin was purified by Ni-NTA (Qiagen) chromatography in a single step.

(11) The optimal quantity of acetolactate required to achieve near complete incorporation in the overexpressed protein was assessed in a series of cultures (90 mL each) in which different amounts of unlabeled precursor were added 1 hour prior induction to final concentrations of 0, 100, 200, 300 and 800 mg/L. The level of incorporation into the purified protein was monitored by directly-detected .sup.13C 1D NMR. When the precursor is incorporated into the overexpressed protein, the .sup.13C-L,V residues are replaced by amino acids with .sup.12C side chains. The quantification was performed by comparing the integral of signals of 4 isolated Leu/Val methyl resonances (19-21 ppm) with respect to the signals of the methyl groups of Ile, Ala (between 9-19 ppm). The addition of 300 mg of pure acetolactate per liter of M9/D.sub.2O culture medium achieves an incorporation level of 95% in Leu/Val side chains without detectable scrambling to other amino-acid biogenesis pathways

(12) Production of U[.sup.2H], U[.sup.15N], Leu/Val-[.sup.13C.sup.1H.sub.3].sup.proS Proteins.

(13) E. coli BL21(DE3) carrying the plasmid of the overexpressed protein (TET2 or MSG) were progressively adapted, in three stages, over 24 h, to M9/D.sub.2O media containing 1 g/L .sup.15ND.sub.4Cl and 2 g/L D-glucose-d.sub.7 (Isotec). In the final culture, the bacteria were grown at 37 C. in M9 media prepared with 99.85% D.sub.2O (Eurisotop). When the O.D. (600 nm) reached 0.8, a solution containing 2-(.sup.13C)methyl-4-(.sup.2H.sub.3)-acetolactate (compound of formula 4) (prepared with the protocol described above) was added. Acetolactate was added to the culture medium to a final concentration of 300 mg/L. 1 hour later, TET2 (/MSG) expression was induced by the addition of IPTG to a final concentration of 1 mM (/0.1 mM). Expression was performed for 3 hours (/12 hours) at 37 C. (/20 C.) before harvesting. For MSG, .sup.13C spectra were recorded at 37 C. in D.sub.2O on a NMR spectrometer operating at a proton frequency of 600 MHz. Only signals for Leu and Val methyl carbons were observed in .sup.13C spectra, indicating that .sup.13C.sup.1H.sub.3 groups of acetolactate were not incorporated in metabolic pathway of other amino-acids.

(14) Production of U[.sup.2H], U[.sup.15N], Ile-[.sup.13C.sup.1H.sub.3].sup.2 Proteins.

(15) E. coli BL21(DE3) carrying the plasmid of the overexpressed protein (TET2 or MSG) were progressively adapted, in three stages, over 24 h, to M9/D.sub.2O media containing 1 g/L .sup.15ND.sub.4Cl and 2 g/L D-glucose-d.sub.7 (Isotec). In the final culture, the bacteria were grown at 37 C. in M9 media prepared with 99.85% D.sub.2O (Eurisotop). When the O.D. (600 nm) reached 0.8, a solution containing 2-(.sup.2H.sub.5)ethyl-2-hydroxy-3-oxo-4(.sup.13C)butanoate (compound of formula 6) (prepared with the protocol described above) was added. Product was added to the culture medium to a final concentration of 300 mg/L. 1 hour later, TET2 (/MSG) expression was induced by the addition of IPTG to a final concentration of 1 mM (/0.1 mM). Expression was performed for 3 hours (/12 hours) at 37 C. (/20 C.) before harvesting.

(16) Production of U[.sup.2H], U[.sup.15N], Leu/Val-[.sup.13C.sup.1H.sub.3, .sup.12C.sup.2H.sub.3] Proteins.

(17) For aim of comparison, the production of perdeuterated proteins with non-stereospecific .sup.13C.sup.1H labeling of Leu and Val methyl groups was achieved using the protocol used before the invention, i.e. the protocol described by V. Tugarinov et al., J. Biomol. NMR 2004, 28, 165-172 and R. Lichtenecker et al., J. Am. Chem. Soc. 2004, 126, 5348-5349.

(18) This protocol is the protocol described above but with the addition 1 hour prior induction of 125 mg/L of 3-(.sup.2H.sub.3)methyl-3-(.sup.2H)-4-(.sup.13C)-ketoisovalerate (Isotec) in place of 300 mg/L of labeled acetolactate (compound of formula 4).

(19) Production of U[.sup.2H], U[.sup.15N], U[.sup.12C], U[.sup.13C.sup.1H.sub.3].sup.proS-Leu/Val, U[.sup.13C.sup.1H.sub.3]-Ala Proteins.

(20) The production of perdeuterated proteins with stereospecific .sup.13C.sup.1H labeling of Leu/Val proS methyl groups and Ala-positions was achieved using the general protocol described above but with the addition 1 hour prior induction of 800 mg/L of 2-(S)-2-(.sup.2H)-3-(.sup.13C)-Alanine (CortecNet) together with 300 mg/L of labeled acetolactate (compound of formula 4). A 2D .sup.13C-methyl TROSY spectra was recorded at 37 C. in D.sub.2O on a NMR spectrometer operating at a proton frequency of 800 MHz. Only peaks corresponding to the expected signals of Alanine methyl groups and proS methyl groups of Leu and Val side chains were observed indicating that labeling using acetolactate derivatives does not interference with other methyl labeling processes.

(21) Proteins Purification.

(22) Malate Synthase G (MSG) was purified initially by Chelating Sepharose chromatography (GE Healthcare) followed by gel filtration chromatography (Superdex 200 pg GE Healthcare). Typical final yields after purification were 60-80 mg/L of methyl specific protonated MSG. The concentration of MSG in typical NMR samples was 1 mM in 100% D.sub.2O buffer containing 25 mM MES (pH 7.0 uncorrected), 20 mM MgCl.sub.2, 5 mM DTT. NMR data were acquired at 37 C.

(23) TET2 was purified using two anion exchange chromatography steps (DEAE Sepharose CL-6B, and Resource Q 6 mL, GE Healthcare) followed by gel filtration (Sephacryl S-300 HR, GE Healthcare). Typical final yield after purification was 20 mg/L of methyl specific protonated TET2. Samples prepared in this manner were demonstrated to be fully active (measured by hydrolytic activity using Leu-4-nitroanitide). The final NMR samples of TET consisted of 80 M TET2 dodecamer (1 mM monomer) in 20 mM Tris (pH 7.4 uncorrected), 20 mM NaCl dissolved in 300 L D.sub.2O. NMR data were acquired at 50 C.

3. NMR Spectroscopy

Experimental Details

(24) All .sup.1H and .sup.13C 1D NMR spectra of ubiquitin and MSG were recorded on a Varian DirectDrive spectrometer operating at a proton frequency of 600 MHz equipped with a cryogenic triple resonance pulsed field gradient probehead.

(25) 2D methyl-TROSY spectra were recorded on a Varian DirectDrive spectrometer operating at a proton frequency of 800 MHz equipped with a cryogenic triple resonance pulsed field gradient probehead. The .sup.1H-.sup.13C HMQC of MSG (/TET2) were recorded with 1288 (/780) complex data points in direct dimension (maximum t.sub.2=99 ms (/60 ms)) and 512 (/380) points in carbon dimension (maximum t.sub.1=128 ms (/47 ms)).

(26) The 4D HMQC-NOESY-HMQC experiments were recorded on a Varian DirectDrive spectrometer operating at a proton frequency of 800 MHz equipped with a cryogenic triple resonance pulsed field gradient probehead. Data were acquired in 96 h of a 1 mM sample of MSG with a NOE mixing time of 300 ms. The experiments were collected with 20 complex points in the indirect .sup.1H dimension (maximum t.sub.1=30 ms), 36 and 18 complex points in the first and second carbon dimension (maximum t.sub.2=21 ms & t.sub.3=11 ms), and 201 complex points in the direct dimension (maximum t.sub.4=80 ms) and 4 scans per increment. All data were processed and analyzed using nmrPipe/nmrDraw and NMRView. Distances were quantified using a full relaxation matrix analysis of NOEs between remote protons in methyl-specific protonated proteins as described in Sounier et al., J. Am. Chem. Soc. 2007, 129, 472-473.

(27) Comparison of Methyl-TROSY Spectra Recorded on Leu/Val Methyl-Specifically Labeled TET2 Samples (468 kDa).

(28) 2D .sup.13C-methyl TROSY spectra were recorded at 50 C. in D.sub.2O on a NMR spectrometer operating at a proton frequency of 800 MHz for U[.sup.2H], U[.sup.12C], Leu/Val-[.sup.12C.sup.2H.sub.3, .sup.13C.sup.1CH.sub.3] TET2 with non-stereospecific [.sup.13C.sup.1H.sub.3]-methyl labeling prepared using 3-(.sup.2H.sub.3)methyl-3-(.sup.2H)-4-(.sup.13C)-ketoisovalerate; and for U[.sup.2H], U[.sup.12C], Leu/Val-[.sup.13C.sup.1H.sub.3].sup.proS TET2 with stereospecific labeling prepared using 2-(.sup.13C)methyl-4-(.sup.2H.sub.3)-acetolactate (compound of formula 4). Preparation of TET2 assembly using a non-stereoselectively labeling scheme results in spectra with substantial cross-peak overlap that would have greatly hampered the observation of amastatin-induced chemical shift changes (Amastatin is an inhibitor of TET2).