Labeled chiral alpha-hydroxy ketoacid derivatives, a process for preparing said derivatives and their use

Abstract

The present invention relates to labeled chiral alpha-hydroxy ketoacid derivatives, a process for preparing the derivatives and their use for isotopic labeling of amino acids, in particular, for isotopic labeling of methyl groups of amino acids, and more particularly, for specific isotopic labeling of valine, leucine and isoleucine methyl groups, in proteins and biomolecular assemblies. The invention also concerns a process for analyzing proteins and biomolecular assemblies by NMR spectroscopy including a step of isotopic labeling of amino acids, in particular, valine, leucine and isoleucine, in proteins and biomolecular assemblies to be analyzed by the chiral alpha-hydroxy ketoacid derivatives of the invention. The invention further relates to a kit for isotopic labeling of valine, leucine and isoleucine amino acids, in proteins and biomolecular assemblies including one or more chiral alpha-hydroxy ketoacid derivatives of the invention.

Claims

1. A composition consisting essentially of a compound of formula (I) having an (S) configuration: ##STR00004## wherein X.sup.1 and X.sup.2 are, independently from each other, .sup.1H (H) or .sup.2H (D); Y.sup.1, Y.sup.2 and Y.sup.3 are, independently from each other, .sup.12C (C) or .sup.13C; R.sup.1 is a methyl group in which the carbon atom is .sup.12C (C) or .sup.13C and the hydrogen atoms are, independently from each other, .sup.1H (H) or .sup.2H (D); R.sup.2 is a methyl group in which the carbon atom is .sup.12C (C) or .sup.13C and the hydrogen atoms are, independently from each other, .sup.1H (H) or .sup.2H (D); or R.sup.2 is an ethyl group in which the carbon atoms are, independently from each other, .sup.12C (C) or .sup.13C and the hydrogen atoms are, independently from each other, .sup.1H (H) or .sup.2H (D), with the proviso that in compound of formula (I), at the same time, at least one hydrogen atom is .sup.2H (D) and at least one carbon atom is .sup.13C.

2. The composition of claim 1, wherein in the compound of formula (I) R.sup.1 is chosen from a group consisting of CD.sub.3, .sup.13CH.sub.3, .sup.13CH.sub.2D and .sup.13CHD.sub.2; R.sup.2 is a methyl group chosen from a group consisting of .sup.13CD.sub.3; or R.sup.2 is an ethyl group chosen from a group consisting of CD.sub.3-CD.sub.2, .sup.13CH.sub.3-CD.sub.2, CH.sub.3—.sup.13CD.sub.2, .sup.13CHD.sub.2-CD.sub.2, .sup.13CHD.sub.2-.sup.13CD.sub.2, .sup.13CH.sub.2D-CD.sub.2, .sup.13CH.sub.2D-.sup.13CD.sub.2 and .sup.13CH.sub.3—.sup.13CD.sub.2.

3. The composition of claim 1, wherein in the compound of formula (I) Y.sup.1═Y.sup.2═Y.sup.3═C.

4. The composition of claim 1, wherein in the compound of formula (I) Y.sup.1═Y.sup.2═Y.sup.3═.sup.13C.

5. The composition of claim 1, wherein said compound of formula (I) is chosen from the group consisting of: (S)-2-(1′-.sup.2H2, 2′-.sup.13C)ethyl-2-hydroxy-3-oxo-4-(.sup.2H.sub.3) butanoic acid {X.sup.1═X.sup.2═H; Y.sup.1═Y.sup.2═Y.sup.3═C; R.sup.1═CD.sub.3; R.sup.2═.sup.13CH.sub.3—CD.sub.2}; (S)-2-(1′-.sup.2H2, 2′-.sup.2H, 2′-.sup.13C)ethyl-2-hydroxy-3-oxo-4-(.sup.2H.sub.3)butanoic acid {X.sup.1═X.sup.2═H; Y.sup.1═Y.sup.2═Y.sup.3═C; R.sup.1═CD.sub.3; R.sup.2═.sup.13CH.sub.2D.sub.−CD.sub.2}; (S)-2-(1′-.sup.2H.sub.2, 2′-.sup.2H.sub.2, 2′-.sup.13C,)ethyl-2-hydroxy-3-oxo-4-(.sup.2H.sub.3)butanoic acid {X.sup.1═X.sup.2═H; Y.sup.1═Y.sup.2═Y.sup.3═C; R.sup.1═CD.sub.3; R.sup.2═.sup.13CHD.sub.2−CD.sub.2}; (S)-2-hydroxy-2-(.sup.2H.sub.3,.sup.13C)methyl-3-oxo-4(.sup.13C)butanoic acid {X.sup.1═X.sup.2═H; Y.sup.1═Y.sup.2═Y.sup.3═C; R.sup.1═.sup.13CH.sub.3; R.sup.2═.sup.13CD.sub.3}; (S)-2-(.sup.2H5)ethyl-2-hydroxy-3-oxo-4-(.sup.13C)methylbutanoic acid {X.sup.1═X.sup.2═H; Y.sup.1═Y.sup.2═Y.sup.3═C; R.sup.1═.sup.13CH.sub.3; R.sup.2═CD.sub.3−CD.sub.2}.

6. The composition of claim 1, wherein said compound of formula (I) is chosen from the group consisting of: (S)-1,2,3-(.sup.13C)-2-(1′-.sup.2H2, .sup.13C2)ethyl-2-hydroxy-3-oxo-4-(.sup.2H3)butanoic acid {X.sup.1═X.sup.2═H; Y.sup.1═Y.sup.2═Y.sup.3═.sup.13C; R.sup.1═CD.sub.3; R.sup.2═CH.sub.3−.sup.13CD.sub.2}; (S)-1,2,3-(.sup.13C)-2-(1′-.sup.2H2, 2′-.sup.2H, .sup.13C.sub.2)ethyl-2-hydroxy-3-oxo-4-(.sup.2H3)butanoic acid {X.sup.1═X.sup.2═H; Y.sup.1═Y.sup.2═Y.sup.3═.sup.13C; R.sup.1═CD.sub.3; R.sup.2═.sup.13CH.sub.2D-.sup.13CD.sub.2}; (S)-1,2,3-(.sup.13C)-2-(1′-.sup.2H2, 2′-.sup.2H2, .sup.13C.sub.2)ethyl-2-hydroxy-3-oxo-4-(.sup.2H.sub.3)butanoic acid {X.sup.1═X.sup.2═H; Y.sup.1═Y.sup.2═Y.sup.3═.sup.13C; R.sup.1═CD.sub.3; R.sup.2═.sup.13CHD.sub.2−.sup.13CD.sub.2}.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Other advantages and features of the present invention may be better understood with respect to the following examples given for illustrative purposes and the accompanying figures.

(2) FIG. 1 represents the enzymatic synthesis of 2-(S)-2-hydroxy,2-ethyl,3-oxobutanoate and 2-(S)-2-hydroxy,2-methyl,3-oxobutanoate (also referred to as precursors) by acetohydroxyacid synthase II (ASAH II) of E. coli. The carbon atoms of the final compound originating from one of the pyruvate molecules are shown in bold.

(3) FIG. 2 represents the enzymatic synthesis of 2-(S)-2-hydroxy,2-ethyl,3-oxobutanoate (also referred to as precursor). Left: one-dimensional NMR spectra of a 1/1 mixture of oxobutanoate/pyruvate in presence of AHAS II as a function of time is represented. Right: the evolution as a function of time of the resonance intensity of the methyl of group of oxobutanoate and the methyl group in position delta-1 of the precursor is represented.

(4) FIG. 3 represents the synthesis of 2-(S)-2-hydroxy,2-(2′-.sup.13C-1′-D.sub.2)-ethyl,3-oxo-4-D.sub.3-butanoate (I).

(5) Upper panel: the NMR spectra of the equimolar mixture of (U-D)-pyruvate and 4-.sup.13C-3-D.sub.2-2-oxobutanoate (left) and the 2-(S)-2-hydroxy,2-(2′-.sup.13C-1′-D.sub.2)-ethyl,3-oxo-4-D.sub.3-butanoate (also referred to as precursor delta-1) after the addition of AHAS II (right) are represented. Some acetolactate (less than 10%) are formed during the reaction but are not observable as they are deuterated.
Lower panel: the synthetic scheme of 2-(S)-2-hydroxy,2-(2′-.sup.13C-1′-D.sub.2)-ethyl,3-oxo-4-D.sub.3-butanoate is represented. .sup.13C labeled carbon atoms are displayed in bold.

(6) FIG. 4 represents the synthesis of 2-(S)-2-hydroxy,2-(D.sub.5)ethyl,3-oxo,4-.sup.13C-butanoate.

(7) Upper panel: the NMR spectra of the equimolar mixture of (U-D)-2-oxobutanoate and 3-.sup.13C-pyruvate (left) and the final product (2-(S)-2-hydroxy,2-(D.sub.5)ethyl,3-oxo,4-.sup.13C-butanoate) after addition of AHAS II (right) are represented. The peaks at 1.35 and 1.57 ppm correspond to the hydrated form of pyruvate (left) and those at 1.32 and 1.55 ppm to acetolactate formed during the reaction (right)
Lower panel: the synthetic scheme of 2-(S)-2-hydroxy,2-(D.sub.5)ethyl,3-oxo,4-.sup.13C-butanoate (also referred to as precursor gamma-2) is represented. .sup.13C labeled carbon positions are displayed in bold.

(8) FIG. 5 represents the synthesis of 2-(S)-2-hydroxy,2-(.sup.13C)methyl,3-oxo,4-.sup.13C-butanoate.

(9) Upper panel: the NMR spectra of the 3-.sup.13C-pyruvate (left) and the final product (2-(S)-2-hydroxy,2-(.sup.13C)methyl,3-oxo,4-.sup.13C-butanoate) after addition of AHAS II (right) are represented. The peaks at 1.35 and 1.57 ppm correspond to the hydrated form of pyruvate.
Lower panel: the synthetic scheme of 2-(S)-2-hydroxy,2-(.sup.13C)methyl,3-oxo,4-.sup.13C-butanoate (also referred to as acetolactate) is represented. .sup.13C labeled carbon positions are displayed in bold.

(10) FIG. 5a represents the synthesis of 2-(S)-2-hydroxy,2-(.sup.13C.sub.2-1′-D.sub.2)-ethyl-3-oxo-1,2,3-.sup.13C-4-D.sub.3-butanoate.

(11) Upper panel: the NMR spectra of the equimolar mixture of (U—.sup.13C)-3-D.sub.2-2-oxobutanoate and (U-D)-2-.sup.13C-pyruvate (left) and the final product after addition of AHAS II (right) are represented. The peaks at 1.35 and 1.57 ppm correspond to the hydrated form of pyruvate (left) and those at 1.32 and 1.55 ppm to acetolactate formed during the reaction (right).
Lower panel: the synthetic scheme of the synthesis of 2-(S)-2-hydroxy,2-(.sup.13C.sub.2-1′-D.sub.2)-ethyl-3-oxo-1,2,3-.sup.13C-4-D.sub.3-butanoate is represented. .sup.13C labeled carbon positions are displayed in bold.

(12) FIG. 6 represents the level of incorporation of (S)-2-hydroxy-2-ethyl-3-oxobutanoate in overexpressed proteins as a function of the amount of exogeneous precursor added. Ubiquitin was expressed in E. coli in M9/D.sub.2O culture medium with 2 g/L of U—[.sup.2H]-glucose. The level of incorporation of (S)-2-hydroxy-2-ethyl-3-oxobutanoate (squares) is compared to those (black circles) of racemic 2-hydroxy-2-[.sup.2H5]ethyl-[1,2,3,4-.sup.13C4]-3-oxobutanoate obtained by chemical synthesis (Ayala et al, 2012, Chem Commun, 48(10):1434-6.). Quantification was performed by comparing the intensities of signals corresponding to isoleucine gamma-2 methyl groups with respect to signals of the epsilon methyl groups of methionine. A level of incorporation in Ile side chains of 95% is obtained by adding about 50 mg of (S)-2-hydroxy-2-ethyl-3-oxobutanoate per liter of M9/D.sub.2O culture medium compared to more than 100 mg per liter for the chemically synthetised precursor

(13) FIG. 7 represents the .sup.13C-HSQC spectra of ubiquitin produced in E. coli either in the presence of labeled alanine and 2-oxobutanoate (upper panel) or in the presence of labeled alanine and the precursor synthetized using the protocol described above (lower panel). The spectra are plotted at 10% of the maximal intensity of the resonances of isoleucine amino acids except within the dotted area where the spectra are plotted at 1%. Using the protocol of Godoy-Ruiz R. et al. (J. Am. Chem. Soc., 2010, 132(51), p. 18340-50) leads to an artifactual labeling of the gamma-2 methyl group of isoleucine of 1.5-2% (upper panel), labeling which is totally suppressed when using our precursors for the culture (lower panel).

(14) FIG. 8 represents the .sup.13C-HSQC spectrum (methyl region) of ubiquitin produced following our labeling protocol as set forth in examples. It can be observed that only proS methyl groups of leucine and valine and the delta-1 methyl groups of isoleucine amino acids are labeled. No leakage to other methyl positions is observed.

DETAILED DESCRIPTION

Examples

Materials and Procedures

(15) The compounds used are commercially available: CDN Isotopes Inc. for (D.sub.5)-2-oxobutanoate, Sigma-Aldrich for 4-(.sup.13C)-2-oxobutanoate, 3-.sup.13C-pyruvate, 2-.sup.13C-pyruvate, pyruvate and Cambridge Isotopes Laboratories for U—(.sup.13C)-2-oxobutanoate. The plasmid carrying the sequences encoding the two subunits of AHAS II was kindly provided by Dr. David Chipman (Ben Gurion University of Negev).

(16) All .sup.1H and .sup.13C one-dimensional NMR spectra were recorded on a Varian DirectDrive spectrometer operating at a proton frequency of 600 MHz equipped with a cryogenic triple resonance pulsed field gradient probe head.

(17) The two-dimensional .sup.1H-.sup.13C HMQC of labeled protein 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)).

Example 1: Preparation of l'acetohydroxyacid synthase II (AHAS II)

(18) The overexpression and purification of AHAS II were made according to the method of Hill et al. (Biochem J. 1997). E. coli BL21 (DE3), carrying the plasmid of the overexpressed AHAS II, were grown at 37° C. in a Luria-Bertani medium.

(19) When the optical density (OD) or absorbance at 600 nm reached 0.5-0.7, AHAS II expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM. Expression was performed for 12 hours at 20° C. The bacteria were harvested by centrifugation at 5000 g for 15 minutes at 4° C., resuspended in 10 ml of TRIS-HCl 0.1 M pH 7.5 and centrifuged at 4000 g for 15 minutes at 4° C. The bacteria were resuspended in 10 ml of buffer (buffer A: TRIS 50 mM pH 8, KCl 0.5 M, imidazole 10 mM and FAD 20 μM). The cells were disrupted by sonication for 2 minutes and the insoluble materials were removed by ultracentrifugation at 45,000 g for 45 minutes at 4° C.

(20) The supernatant was then deposed on a NiNTA column (obtained from Qiagen) equilibrated with buffer A. After washing the column with 5 volumes of buffer A, the AHAS II was eluted using buffer B (buffer B: TRIS 50 mM pH 8, KCl 0.5 M, imidazole 400 mM and FAD 20 μM). The fractions containing AHAS II were pooled, concentrated, dialyzed against deionized and lyophilized. The activity of AHAS II was determined by measuring the decrease of absorbance of pyruvate at 333 nm.

Example 2: Synthesis of 13C and 2H (D) labeled 2-(S)-2-hydroxy,2-ethyl,3-oxo,4-butanoate and 2-(S)-2-hydroxy,2-methyl,3-oxo,4-butanoate

(21) The synthesis two compounds according to the invention: 2-(S)-2-hydroxy,2-ethyl,3-oxo,4-butanoate and 2-(S)-2-hydroxy,2-methyl,3-oxo,4-butanoate labeled with D and .sup.13C, was performed according to the protocol described by D. Chipman for the chiral synthesis of aromatic alpha-hydroxy ketones (Biotechnol Bioeng. 2004 88(7):825-31 and US 2006/0148042).

(22) Synthesis of the .sup.13C and .sup.2H labeled compounds was made by adding an aliquot of AHAS II, purified as described above, in an equimolar mixture of .sup.13C and .sup.2H (D) labeled pyruvate and 2-oxobutanoate. The reaction was monitored by .sup.1H NMR spectra (one-dimensional) and condensation of pyruvate with 2-oxobutanoate was completed after 2 hours as shown in FIG. 2. A large number of compounds of formula (I) can be synthesized enzymatically using reagents differently labeled by .sup.13C and .sup.2H.

A. Synthesis of 2-(S)-2-hydroxy,2-(2′-13C-1′-D2)-ethyl,3-oxo-4-D3-butanoate (precursor delta-1)

(23) 33 mM of (U-D)-pyruvate (perdeuterated by treatment of unlabeled pyruvate in D.sub.2O at pH 10.7 for 72 hours) was mixed with 33 mM of 4-.sup.13C,3-D.sub.2-2-oxobutanoate in 3 ml of D.sub.2O buffer potassium phosphate 50 mM pH 7.8, MgCl.sub.2 10 mM, Thiamine diphosphate 1 mM, FAD 20 mM.

(24) The reaction was initiated by the addition of 6 mM (420 mg/ml) of AHAS II and followed by .sup.1H NMR (one-dimensional). .sup.1H NMR Spectra of initial and final compounds are represented in FIG. 3.

B. Synthesis of 2-(S)-2-hydroxy,2-(D5)ethyl,3-oxo,4-13C-butanoate (precursor gamma-2)

(25) 33 mM of 3-.sup.13C-Pyruvate was mixed with the same concentration of D.sub.5-2-oxobutanoate in 3 ml of D.sub.2O buffer potassium phosphate 50 mM pH 7.8, MgCl.sub.2 10 mM, Thiamine diphosphate 1 mM, FAD 20 mM.

(26) The reaction was initiated by the addition of at 6 mM (420 mg/ml) of AHAS II and followed by .sup.1H NMR (one-dimensional). .sup.1H NMR Spectra of initial and final compounds are presented in FIG. 4.

C. Synthesis of 2-(S)-2-hydroxy,2-(13C)methyl,3-oxo,4-(13CD3)-butanoate (acetolactate)

(27) 66 mM of 3-.sup.13C-pyruvate was dissolved in 3 ml of D.sub.2O buffer potassium phosphate 50 mM pH 7.8, MgCl.sub.2 10 mM, Thiamine diphosphate 1 mM, FAD 20 mM.

(28) The reaction was initiated by the addition of 6 mM (420 mg/ml) of AHAS II and followed by .sup.1H NMR (one-dimensional). .sup.1H NMR spectra of pyruvate and final compound (2-(S)-2-hydroxy,2-(.sup.13C)methyl,3-oxo,4-(.sup.13CD.sub.3)-butanoate) are presented in FIG. 5. Deuteration at position 4 is accomplished by .sup.1H/.sup.2H(D) exchange as described by P. Gans et al. (Angew. Chem. Int. Ed., 2010, 49, pages 1958-1962).

D. Synthesis of 2-(S)-2-hydroxy,2-(13C2-1′-D2)-ethyl-3-oxo-1,2,3-13C-4-D3-butanoate

(29) 33 mM of U-D, 2-.sup.13C-Pyruvate was mixed with U—.sup.13C,3-D.sub.2-2-oxobutanoate at the same concentration in 3 ml of D.sub.2O buffer potassium phosphate 50 mM pH 7.8, MgCl.sub.2 10 mM, Thiamine diphosphate 1 mM, FAD 20 mM.

(30) The reaction was initiated by the addition of 6 mM (420 mg/ml) of AHAS II and followed by .sup.1H NMR (one-dimensional). .sup.1H NMR Spectra of initial and final compounds are presented in FIG. 5a.

Example 3: Optimization of the Incorporation of (S)-2-hydroxy-2-ethyl-3-oxobutanoate in Overexpressed Protein

(31) Initial experiments to determine the level of (S)-2-hydroxy-2-ethyl-3-oxobutanoate incorporation into overexpressed proteins were performed using ubiquitin as a model system.

(32) E. coli BL21(DE3) cells were transformed with a pET41c plasmid (obtained from Novagen) carrying the human His-tagged ubiquitin gene (pET41c-His-Ubi) and transformants were grown in M9/D.sub.2O media containing 1 g/L of .sup.15ND.sub.4Cl and 2 g/L of U—[.sup.2H]-glucose.

(33) When the optical density or absorbance at 600 nm reached 0.8, a solution containing labeled 2-hydroxy-2-ethyl-3-oxobutanoate was added. After an additional 1 hour, ubiquitin expression was induced by the addition of β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Induction was performed for 3 hours at 37° C. Ubiquitin was purified by Ni-NTA (obtained from Qiagen) chromatography column in a single step.

(34) The optimal quantity of 2-hydroxy-2-ethyl-3-oxobutanoate required to achieve almost complete incorporation in the overexpressed protein was assessed in a series of cultures (100 mL each) in which different amounts of labeled precursor were added 1 hour prior induction, to final concentrations of 15, 30, 60, 80 and 100 mg/L together with 200 mg/mL U—[.sup.13C]-methionine. The level of incorporation into the purified protein was monitored by .sup.13C-HSQC NMR. The quantification was performed by comparing the integral of signals corresponding to Ile methyl groups with respect to the signals of the epsilon methyl groups of Met.

(35) The addition of about 50 mg of pure (S)-2-hydroxy-2-ethyl-3-oxobutanoate per liter of M9/D.sub.2O culture medium achieves an incorporation level of 95% in Ile side chains (FIG. 6).

Example 4: Production of U—[2H], U—[15N], Ile-d1-[13C1H3] proteins

(36) E. coli BL21(DE3) cells, transformed with a pET41c plasmid carrying the human His-tagged ubiquitin gene (pET41c-His-Ubi), were progressively adapted, in three stages, over 24 hours, to a M9/D.sub.2O medium containing 1 g/L .sup.15ND.sub.4Cl and 2 g/L D-glucose-d.sub.7 (obtained from Isotec). In the final culture, the bacteria were grown at 37° C. in a M9 medium prepared with 99.85% D.sub.2O (obtained from Eurisotop).

(37) When the optical density or absorbance at 600 nm reached 0.8, a solution containing (S)-2-hydroxy,2-[2′-.sup.13C,1′-D.sub.2]ethyl,[4-D.sub.3]-3-oxo-butanoate (prepared according to the protocol described in example 2-A) and 2-oxoisovalerate-d.sub.7 was added to the culture medium to a final concentration of 65 mg/L and 200 mg/L, respectively. 1 hour later, ubiquitin expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Expression was performed for 3 hours at 37° C. before harvesting. Ubiquitin was purified by Ni-NTA (obtained from Qiagen) column chromatography in a single step.

(38) .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 delta-1 isoleucine methyl carbons were observed in .sup.13C spectra indicating that .sup.13C.sup.1H.sub.3 groups of (S)-2-hydroxy,2-[2-.sup.13C,1-D.sub.2]ethyl,[4-D.sub.3]-3-oxo-butanoate were not incorporated in the metabolic pathways of other amino acids. The incorporation level of .sup.13CH.sub.3 groups in the delta-1 position of the isoleucine amino acids was estimated to be higher than 95% based on the integration NMR signals observed in a two-dimensional .sup.1H—.sup.13C HMQC of labeled the protein.

Example 5: Production of U—[2H], U—[5N], Ile-g2-[13C1H3] proteins

(39) E. coli BL21(DE3) cells, transformed with a pET41c plasmid carrying the human His-tagged ubiquitin gene (pET41c-His-Ubi), were progressively adapted, in three stages, over 24 hours, to a M9/D.sub.2O medium containing 1 g/L .sup.15ND.sub.4Cl and 2 g/L D-glucose-d.sub.7 (obtained from Isotec). In the final culture, the bacteria were grown at 37° C. in a M9 medium prepared with 99.85% D.sub.2O (obtained from. Eurisotop).

(40) When the optical density or absorbance at 600 nm reached 0.8, a solution containing (2-(S)-2-hydroxy,2-(D.sub.5)ethyl,3-oxo,4-.sup.13C-butanoate (prepared according to the protocol described in example 2-B) and 2-oxoisovalerate-d.sub.7 was added to the culture medium to a final concentration of 65 mg/L and 200 mg/L respectively. 1 hour later, ubiquitin expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Expression was performed for 3 hours at 37° C. before harvesting. Ubiquitin was purified by Ni-NTA (obtained from Qiagen) column chromatography in a single step.

(41) .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 the gamma-2 methyl carbons of isoleucine were observed in .sup.13C spectra, indicating that .sup.13C.sup.1H.sub.3 groups of 2-(S)-2-hydroxy,2-(D.sub.5)ethyl,3-oxo,4-.sup.13C-butanoate were not incorporated in metabolic pathways of other amino acids. The incorporation level of .sup.13CH.sub.3 groups in the gamma-2 position of isoleucine was estimated to be higher than 95% based on the integration NMR signals observed in a two-dimensional .sup.1H—.sup.13C HMQC of labeled protein.

Example 6: Production of U—[2H], U—[15N], Ala-beta[13C1H], Ile-delta-1-[13C1H3] proteins using the protocol of Godoy-Ruiz et al. (J. Am. Chem. Soc. 2010, 132, pages 1834048350)

(42) E. coli BL21(DE3) cells, transformed with a pET41c plasmid carrying the human His-tagged ubiquitin gene (pET41c-His-Ubi), were progressively adapted, in three stages, over 24 hours, to a M9/D.sub.2O medium containing 1 g/L .sup.15ND.sub.4Cl and 2 g/L D-glucose-d.sub.7 (obtained from Isotec). In the final culture, the bacteria were grown at 37° C. in M9 media prepared with 99.85% D.sub.2O (obtained from Eurisotop), 2.5 g/L of succinate-d.sub.5 (obtained from Isotec).

(43) When the optical density or absorbance at 600 nm reached 0.8, a solution containing .sup.13CH.sub.3-alanine, 4-.sup.13C-2-ketobutyrate and 2-ketoisovalerate-d.sub.7 were added to the culture medium to a final concentration of respectively 800, 75 and 200 mg/L. Note that to simplify spectra and avoid overlap, 4-.sup.13C,3-methyl(.sup.2H.sub.3)-2-ketoisovalerate from original protocol was substituted by perdeuterated forms of the precursors. 1 hour later, ubiquitin expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Expression was performed for 3 hours at 37° C. before harvesting. Ubiquitin was purified by Ni-NTA (obtained from Qiagen) column chromatography in a single step.

(44) .sup.13C spectra were recorded at 37° C. in D.sub.2O on a NMR spectrometer operating at a proton frequency of 600 MHz. Signals for alanine and delta-1 methyl carbons of isoleucine were observed in .sup.13C spectra, but also spurious peaks corresponding to the resonance gamma-2 methyl carbons of isoleucine were observed. The level of isotopic scrambling in gamma-2 methyl carbons of isoleucine was estimated to be 1.5-2% as shown in FIG. 7. The incorporation level of .sup.13CH.sub.3 groups in the delta-1 position of isoleucine and beta position of alanine was estimated to be higher than 95% based on the integration NMR signals observed in a two-dimensional .sup.1H—.sup.13C HMQC of labeled protein.

Example 7: Production of U—[2H], U—[15N], Ala-beta[13C1H3], Ile-delta-1-[13C1H3], Leu/Val-[13C1H3] pro S proteins

(45) E. coli BL21(DE3) cells, transformed with a pET41c plasmid carrying the human His-tagged ubiquitin gene (pET41c-His-Ubi), were progressively adapted, in three stages, over 24 hours, to a M9/D.sub.2O medium containing 1 g/L .sup.15ND.sub.4CI and 2 g/L D-glucose-d.sub.7 (obtained from Isotec). In the final culture, the bacteria were grown at 37° C. in a M9 medium prepared with 99.85% D.sub.2O (obtained from Eurisotop).

(46) When the optical density or absorbance at 600 nm reached 0.8, a solution containing .sup.13CH.sub.3-alanine, 2-(S)-2-hydroxy,2-(2′-.sup.13C-1′-D.sub.2)-ethyl,3-oxo-4-D.sub.3-butanoate and 2-hydroxy,2-(.sup.13C)methyl,3-oxo,4-(.sup.13CD.sub.3)-butanoate precursors was added to the culture medium to a final concentration of respectively 800 mg/L, 65 mg/L, 400 mg/L.

(47) When using 2-(S)-2-hydroxy,2-(.sup.13C)methyl,3-oxo,4-(.sup.13CD.sub.3)-butanoate precursor (prepared enzymatically as described in example 2-C) the amount of the last compound can be reduced to 200 mg/L instead of 400 mg/L. 1 hour later, ubiquitin expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Expression was performed for 3 hours at 37° C. before harvesting. Ubiquitin was purified by Ni-NTA (obtained from Qiagen) column chromatography in a single step.

(48) .sup.13C spectra were recorded at 37° C. in D.sub.2O on a NMR spectrometer operating at a proton frequency of 600 MHz. Signals for Ala, Ile-delta-1, Leu/Val-proS methyl carbons were observed in .sup.13C spectra, without detectable spurious peaks corresponding to resonance of the gamma-2 of isoleucine methyl carbons. The incorporation level of .sup.13CH.sub.3 groups in the gamma-2 position of isoleucine, Leu/Val-proS and the beta position of Ala was estimated to be higher than 95% based on the integration NMR signals observed in a two-dimensional .sup.1H—.sup.13C HMQC of labeled proteins.

(49) It is thus clear that using the protocols described above suppresses the artifacts of labeling, i.e. the residual labeling of the gamma-2 methyl groups of isoleucine that impedes collecting required data for structural determination (NOE), as illustrated in FIG. 7.