Synthesis of [1-¹³C]2-Oxoglutaric Acid and ¹³C Breath Tests Designed to Assess TCA Cycle Flux

Abstract

Several approaches for synthesizing [1-¹³C]2-oxoglutaric acid were attempted, and the synthesis was successfully achieved in 4 steps from trimethylsilyl ¹³C-cyanide. The ¹³C-breath tests on rats were conducted by orally administering the newly synthesized [1-¹³C]2-oxoglutaric acid, the previously prepared [1′-¹³C]citric acid, and [1-¹³C]acetic acid as a control drug, and the results were compared. The results indicate that [1-¹³C]2-oxoglutaric acid and [1′-¹³C]citric acid may serve as potential substrates for assessing the TCA cycle flux.

Introduction

The tricarboxylic acid (TCA) cycle in mitochondria converts nutrients into energy and provides precursors for the biosynthesis of amino acids, sugars, fatty acids, and nucleotides.¹⁾ Mitochondrial functions, including the TCA cycle, are affected by health, disease, and aging.²⁾ In the TCA cycle, CO₂ is generated from the oxidative decarboxylation of d-isocitric acid to form 2-oxoglutaric acid, and from the conversion of 2-oxoglutaric acid to form succinyl-CoA (Chart 1).

Chart 1. Metabolic Conversion of Pyruvic Acid and the TCA Cycle

Breath tests utilizing ¹³C-labeled compounds as probes (¹³C-breath tests) are non-invasive and convenient methods for clinical diagnostic procedures.³⁾ These tests measure the isotope ratio of carbon dioxide (¹³CO₂/¹²CO₂) in exhaled breath, influenced by the metabolic process of a ¹³C-labeled test drug. To date, ¹³C-breath tests utilizing [1-¹³C]pyruvic acid (¹³C-PA) and [1-¹³C]acetic acid (¹³C-AA) have been reported for assessing mitochondrial function.⁴⁾ However, their reported method is ineffective for assessing TCA cycle flux because the ¹³C-PA metabolic pathway branches into 2 routes: one produces [1-¹³C]oxaloacetate by pyruvate carboxylase, and the other generates acetyl-CoA by pyruvate dehydrogenase complex. The conversion of ¹³C-PA to acetyl-CoA results in the loss of the ¹³C label before entering the TCA cycle. Additionally, ¹³C-AA is converted into ¹³C-acetyl-CoA by acetyl-CoA synthetase, then enters the TCA cycle and is selectively transformed into [5-¹³C]2-oxoglutaric acid.^5,6) In the conversion of 2-oxoglutaric acid to succinyl-CoA, ¹³CO₂ is generated only from [1-¹³C]2-oxoglutaric acid (¹³C-2OG). On the other hand, the 1′-carboxy group of citric acid is converted into CO₂ via ¹³C-d-isocitric acid in the TCA cycle. Therefore, we developed a method for preparing [1′-¹³C]citric acid (¹³C-CA) and obtained preliminary results of a ¹³C-breath test on rats by administering it.⁷⁾

In this paper, we developed a method for synthesizing ¹³C-2OG, which is expected to serve as an effective ¹³C-probe to assess the metabolism that forms succinyl-CoA in the TCA cycle. Although ¹³C-2OG is commercially available, it is expensive and difficult to obtain in sufficient quantities for ¹³C-breath test studies. Thus, a scalable synthetic method for ¹³C-2OG is essential. We also conducted ¹³C-breath tests in rats, administering ¹³C-2OG, ¹³C-CA, and ¹³C-AA as probe molecules, and compared the time course of the increase in the ratios of ¹³CO₂/¹²CO₂ (Δ¹³CO₂) exhaled in the breath.

Results and Discussion

Several synthetic routes for ¹³C-2OG were tested using unlabeled reagents before utilizing ¹³C-labeled ones. We initially intended to obtain ¹³C-2OG by hydrolyzing the corresponding 4-([¹³C]cyano)-4-oxobutanoic acid derivative formed by the reaction of the ¹³C-cyanide ion.⁸⁾ However, the desired 4-cyano-4-oxobutanoic acid could not be obtained by adding potassium cyanide or tetraethylammonium cyanide to succinic anhydride (Chart 2). Therefore, methyl 4-cyano-4-oxobutanoate was synthesized in 3 steps: 1) methanolysis of succinic anhydride, 2) conversion of the remaining carboxy group to the acid chloride using thionyl chloride, and 3) the cyano substitution reaction using cuprous cyanide.⁹⁾ However, neither the hydrolysis nor the methanolysis of the cyano group in methyl 4-cyano-4-oxobutanoate succeeded when using hydrochloric acid or methanol–chlorotrimethylsilane, respectively.

Chart 2. The Initial Synthetic Effort of 2-Oxoglutaric Acid Derivatives

In this solvolysis, the carbonyl group at the γ-position may have facilitated the elimination of the cyano group, leading to succinic acid or dimethyl succinate (Chart 3).

Chart 3. The Mechanism of Solvolysis for Methyl 4-Cyano-4-oxobutanoate

We designed an alternative synthetic route for ¹³C-2OG using 4-hydroxy amides as starting materials and intermediates, since amide carbonyls are more resistant to nucleophilic attack by the intramolecular hydroxyl group to form lactones than ester carbonyls. In this route, the formation of ¹³C-cyanohydrin trimethylsilyl ether was chosen for ¹³C labeling (Chart 4).

Chart 4. The Synthetic Route Toward ¹³C-2OG via 4-Hydroxy Amides

Amides used as candidates of a starting material were obtained by the aminolysis of γ-butyrolactone using pyrrolidine,¹⁰⁾ piperidine, morpholine,¹¹⁾ and diethylamine,¹²⁾ respectively. Considering large-scale synthesis, catalytic oxidation using 2-azaadamantane N-oxyl (AZADO) with sodium hypochlorite pentahydrate as a reoxidizer was consistently chosen for this synthetic route.¹³⁾ The reactions of the aldehydes and trimethylsilyl cyanides to form cyanohydrin trimethylsilyl ethers were performed in the presence of trimethylamine as a catalyst.¹⁴⁾ The methanolysis of the cyano group was conducted with chlorotrimethylsilane in methanol to obtain compound A. Since the amides were relatively unstable during work-up and handling, these reactions to compound A were thus carried out continuously.

As shown in Table 1, the best yields of compounds A and B were obtained when diethylamide was used as the starting material (entry 4). The amides derived from cyclic amines (entries 1–3) were easily accessible from γ-butyrolactone, but they exhibited greater instability in subsequent reactions. The amide derived from piperidine (entry 2) gave dimethyl 2-hydroxyglutarate as a by-product, which was readily cyclized to γ-lactone under unneutralized conditions. Furthermore, amides derived from morpholine (entry 3) could not withstand the conditions of AZADO oxidation to afford the desired aldehyde.

Table 1. Yields of Compounds A and B from the Corresponding Amides

Entry	R2N —	Yield (%)
		A (in 3 steps)	B
1		27	40
2		21*	41
3		0	—
4		74	72

*Dimethyl 2-hydroxyglutarate (37%) was obtained as a by-product.

Finally, the amide and ester groups of compound B derived from diethylamide (entry 4) were simultaneously hydrolyzed using 4 M hydrochloric acid to give 2-oxoglutaric acid in a 91% yield. According to the experimental conditions, ¹³C-2OG was successfully obtained from diethylamide as the starting material in a total yield of 54% based on trimethylsilyl ¹³C-cyanide (TMS¹³CN).¹⁵⁾

For now, since the desired ¹³C-2OG was available, the ¹³C-breath tests were conducted by orally administering ¹³C-2OG, the previously prepared ¹³C-CA, and ¹³C-AA to rats to compare the results (Fig. 1). ¹³C-AA was used as a control reagent because it is widely employed in ¹³C-breath tests for gastrointestinal absorption studies and is commercially available.

Fig. 1. The Time Course of the Δ¹³CO₂ (‰) Values after the Oral Administration of ¹³C-Labeled Compounds to Rats (120 μmol/kg)

Data are presented as means with standard error (S.E.) (n = 8). *p < 0.05 (CA vs. 2OG and AA); ^#p < 0.05 (CA vs. 2OG).

For each compound, the Δ¹³CO₂ values in the exhaled air peaked approximately 30 min after administration. The results suggest that these compounds were absorbed and metabolized in a similar manner in the TCA cycle.

Conclusion

The synthesis of ¹³C-2OG was achieved in 4 steps from TMS¹³CN. The ¹³C-breath tests indicate that ¹³C-2OG and ¹³C-CA may serve as potential substrates for assessing the TCA cycle flux. Currently, ¹³C-breath tests using these substrates are being planned for animal disease models in which the TCA cycle flux could be affected.

Experimental

General

Most reagents and solvents used in the synthesis were commercially available and applied without additional purification. Copper(I) cyanide was synthesized from potassium cyanide, copper sulfate pentahydrate, and sodium hydrogen sulfate, according to the reported procedure.^{9) 13}C-CA was synthesized from dimethyl 3-oxo-1,5-pentanedioate and TMS¹³CN by the method previously reported.^{7) 13}C-AA (99 atom% ¹³C) was purchased from Cambridge Isotope Laboratories Inc. (Tewksbury, MA, U.S.A.). TMS¹³CN was prepared from potassium ¹³C-cyanide (99 atom% ¹³C; Cambridge Isotope Laboratories Inc.) according to the procedure reported by Reez and Chatziiosifidis.^{16) 1}H- and ¹³C-NMR spectra were recorded with a Bruker BioSpin AVANCE 500 spectrometer (Bruker BioSpin Corp., Billerica, MA, U.S.A.). Chemical shifts were reported on a δ (ppm) scale with tetramethylsilane as an internal standard (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad). High-resolution electrospray ionization MS (ESIMS) analyses were performed with a Bruker Daltonics micrOTOF-Q spectrometer (Bruker Daltonics, Billerica, MA, U.S.A.).

Succinic Acid Monomethyl Ester

Methanol (10 mL) was added to succinic anhydride (1.5 g, 15 mmol) and was stirred at 70°C for 2 h. The reaction mixture was concentrated under reduced pressure to give succinic acid mono methyl ester (1.9 g, 98% yield). The product was used for the subsequent reaction without further purification. ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 2.63 (2H, m), 2.69 (2H, m), 3.71 (3H, s); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 28.6, 28.8, 51.9, 172.5, 177.5.

Methyl 4-Chloro-4-oxobutanoate

Thionyl chloride (5.0 mL, 69 mmol) was added to the above carboxylic acid (1.9 g, 15 mmol), and the mixture was refluxed for 2 h. The reaction mixture was distilled under reduced pressure to give methyl 4-chloro-4-oxobutanoate (1.68 g, 74% yield); boiling point 93°C (18 mmHg). ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 2.69 (2H, t, J = 6.6), 3.21 (2H, t, J = 6.6), 3.72 (3H, s); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 29.0, 41.7, 52.1, 171.2, 172.9.

Methyl 4-Cyano-4-oxobutanoate

To a suspension of copper(I) cyanide (900 mg, 10 mmol) in acetonitrile was added the above chloride (1.2 mL, 10 mmol), and the mixture was refluxed for 2 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in diethyl ether and filtered through paper. The filtrate was concentrated under reduced pressure, and the residue was dissolved in chloroform. After filtering twice through paper, methyl 4-cyano-4-oxobutanoate (1.4 g, 100% yield) was obtained. ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 2.74 (2H, t, J = 6.4), 3.07 (2H, t, J = 6.4), 3.72 (3H, s); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 27.0, 39.8, 52.3, 113.0, 171.3, 175.2.

4-Hydroxy-1-(pyrrolidin-1-yl)butan-1-one

A mixture of γ-butyrolactone (4.6 mL, 60 mmol) and pyrrolidine (9.9 mL, 120 mmol) in triethylamine (34 mL) was refluxed overnight. The reaction mixture was concentrated under reduced pressure to give 4-hydroxy-1-(pyrrolidin-1-yl)butan-1-one (10 g, 100% yield). The product was used for the subsequent reaction without further purification. ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.87 (2H, quin, J = 7.1), 1.92 (2H, quin, J = 5.4), 1.97 (2H, quin, J = 7.0), 2.46 (2H, t, J = 6.5), 3.43 (2H, t, J = 6.8), 3.47 (2H, t, J = 7.0), 3.70 (2H, t, J = 5.6); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 24.5, 26.2, 27.3, 32.9, 46.0, 46.9, 63.0, 172.4; ESI-TOFMS (pos.) m/z: 180.1002 [M + Na]⁺ (Calcd for C₈H₁₅NNaO₂: 180.0995).

4-Hydroxy-1-(piperidin-1-yl)butan-1-one

A mixture of γ-butyrolactone (10 g, 116 mmol) and piperidine (10 g, 116 mmol) was stirred overnight at 100°C. The reaction mixture was diluted with chloroform and washed with brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 4-hydroxy-1-(piperidin-1-yl)butan-1-one (17 g, 85% yield). The product was used for the subsequent reaction without further purification. ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.60–1.52 (4H, m), 1.64 (2H, m), 1.91 (2H, quin, J = 6.4), 2.50 (2H, t, J = 6.6), 3.16 (1H, m (OH)), 3.42 (2H, t, J = 5.6), 3.56 (2H, t, J = 5.6), 3.70 (2H, m); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 24.6, 25.7, 26.6, 27.8, 31.2, 43.0, 46.9, 62.9, 171.9; ESI-TOFMS (pos.) m/z: 194.1170 [M + Na]⁺ (Calcd for C₉H₁₇NNaO₂: 194.1150).

4-Hydroxy-1-morpholinobutan-1-one

A mixture of γ-butyrolactone (10 g, 116 mmol) and morpholine (10 g, 116 mmol) was stirred overnight at 120°C. The reaction mixture was chromatographed on a silica gel column with chloroform–methanol (9 : 1) to give 4-hydroxy-1-morpholinobutan-1-one (16 g, 81% yield). ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.92 (2H, quin, J = 6.4), 2.49 (2H, t, J = 6.8), 2.81 (1H, t, J = 5.1 (OH)), 3.49 (2H, t, J = 4.7), 3.62 (2H, m), 3.73–3.65 (6H, m); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 27.5, 30.4, 42.0, 46.0, 62.5, 66.6, 66.8,172.1; ESI-TOFMS (pos.) m/z: 196.0937 [M + Na]⁺ (Calcd for C₈H₁₅NNaO₃: 196.0944).

N,N-Diethyl-4-hydroxybutanamide

To a solution of diethylammonium chloride (5.6 g, 51 mmol) in tetrahydrofuran (100 mL) was added 3.0 mol/L diisobutylaluminum hydride in toluene (34 mL, 102 mmol), and the mixture was stirred at room temperature for 2 h under an argon atmosphere. To the reaction mixture was added γ-butylolactone (1.8 mL, 23 mmol), followed by stirring at room temperature for an additional hour. The reaction mixture was diluted with diethyl ether, and a saturated solution of sodium sulfate in water was carefully added with vigorous stirring. After stirring for several minutes, the white solid was separated in the solution. The supernatant was collected, and the residue was washed with diethyl ether. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with chloroform–methanol (19 : 1) to give N,N-diethyl-4-hydroxybutanamide (3.6 g, 98% yield) as a colorless oil. ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.12 (3H, t, J = 7.2), 1.19 (3H, t, J = 7.2), 1.90 (2H, dt, J = 6.5, 6.2), 2.51 (2H, t, J = 6.5), 3.33 (2H, q, J = 7.2), 3.39 (2H, q, J = 7.2), 3.70 (2H, m); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 13.0, 14.2, 27.6, 31.0, 40.5, 42.2, 62.9, 172.8; ESI-TOFMS (pos.) m/z: 182.1173 [M + Na]⁺ (Calcd for C₈H₁₇NNaO₂: 182.1151).

General Synthesis Procedure for Compound A

1) To a solution of the amide (5.0 mmol, 1.0 equivalent [equiv.]) in dichloromethane (0.33 mol/L) was added tetrabutylammonium hydrogensulfate (0.05 equiv,), AZADOL® (0.01 equiv.), and sodium hypochlorite pentahydrate (1.1 equiv.) at 0°C, followed by stirring at room temperature for half an hour. The reaction mixture was diluted with ethyl acetate and washed successively with a saturated solution of sodium bicarbonate in water, water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude aldehyde was used for the subsequent reaction without further purification.

2) To a solution of the above aldehyde in dichloromethane (0.5 mol/L) was added trimethylsilyl cyanide (1.0 equiv.) and triethylamine (0.1 equiv.) at 0°C, followed by stirring at room temperature for one and a half hours. The reaction mixture was diluted with ethyl acetate and washed successively with water and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude cyanohydrin trimethylsilyl ether was used for the subsequent reaction without further purification.

3) To a solution of the above cyanohydrin trimethylsilyl ether in methanol (0.5 mol/L), chlorotrimethylsilane (5 equiv.) was added and stirred at 50°C overnight. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in chloroform and the precipitate was filtered off. The filtrate was concentrated under reduced pressure. The residue was chromatographed on a silica gel column to give compound A.

Methyl 2-Hydroxy-5-oxo-5-(Pyrrolidin-1-yl)pentanoate

4-Hydroxy-1-(pyrrolidin-1-yl)butan-1-one (790 mg) was used as the starting material. The chromatography on a silica gel column with chloroform–methanol (19 : 1) gave methyl 2-hydroxy-5-oxo-5-(pyrrolidin-1-yl)pentanoate (296 mg, 3 steps, 27% yield). ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.86 (2H, quin, J = 6.8), 1.95 (2H, quin, J = 6.8), 2.02 (1H, m), 2.24 (1H, m), 2.47 (2H, m), 3.41 (2H, dt, J = 1.8, 6.9), 3.47 (2H, t, J = 6.9), 3.78 (3H, s), 4.28 (1H, s, −OH), 4.29 (1H, m); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 24.3, 26.0, 28.9, 30.5, 45.8, 46.7, 52.3, 70.4, 171.2, 175.1; ESI-TOFMS (pos.) m/z: 238.1072 [M + Na]⁺ (Calcd for C₁₀H₁₇NNaO₄: 238.1050).

Methyl 2-Hydroxy-5-oxo-5-(Piperidin-1-yl)pentanoate and Dimethyl 2-Hydroxypentanedioate

4-Hydroxy-1-(piperidin-1-yl)butan-1-one (860 mg) was used as the starting material. The chromatography on a silica gel column with chloroform–methanol (19 : 1) gave methyl 2-hydroxy-5-oxo-5-(piperidin-1-yl)pentanoate (240 mg, 3 steps, 21% yield) and dimethyl 2-hydroxypentanedioate (324 mg, 3 steps, 37% yield). Dimethyl 2-hydroxypentanedioate was slowly converted into methyl 5-oxotetrahydrofuran-2-carboxylate. Methyl 2-hydroxy-5-oxo-5-(piperidin-1-yl)pentanoate: ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.54 (4H, m), 1.63 (2H, m), 2.00 (1H, m), 2.23 (1H, m), 2.50 (2H, m), 3.39 (2H, t, J = 5.4), 3.55 (2H, t, J = 5.5), 3.78 (3H, s), 3.90 (1H, m, -OH), 4.28 (1H, m); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 24.5, 25.5, 26.4, 29.0, 29.4, 42.9, 46.6, 52.4, 70.3, 170.7, 175.2; ESI-TOFMS (pos.) m/z: 252.1197 [M + Na]⁺ (Calcd for C₁₁H₁₉NNaO₄: 252.1206). Dimethyl 2-hydroxypentanedioate: ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 4.25 (1H, dd, J = 8.0, 4.5), 3.79 (3H, s), 3.68 (3H, s), 3.01(1H, d, J = 4.5 [OH]), 2.55–2.42 (2H, m), 2.21–2.14 (1H, m), 1.98–1.93(1H, m); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 29.2, 29.3, 51.6, 52.5, 69.4, 173.5, 174.9; ESI-TOFMS (pos.) m/z: 199.0586 [M + Na]⁺ (Calcd for C₇H₁₂NaO₅: 199.0577). Methyl 5-oxotetrahydrofuran-2-carboxylate: ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 4.96 (1H, dd, J = 8.0, 4.5), 3.81 (3H, s), 2.65–2.51 (3H, m), 2.37–2.31 (1H, m); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 25.6, 26.5, 52.6, 75.5, 170.2, 175.8.

Methyl 5-Diethylamino-2-hydroxy-5-oxopentanoate

N,N-Diethyl-4-hydroxybutanamide (800 mg) was used as the starting material. The chromatography on a silica gel column with chloroform–ethyl acetate (19 : 1) gave methyl 5-(diethylamino)-2-hydroxy-5-oxopentanoate (807 mg, 3 steps, 74% yield). ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.12 (3H, t, J = 7.2), 1.18 (3H, t, J = 7.2), 2.01 (1H, m), 2.23 (1H, m), 2.50 (1H, m), 3.31 (2H, q, J = 7.2), 3.37 (2H, dq, J = 1.5, 7.2), 3.78 (3H, s), 4.02 (1H, d, J = 5.4, −OH), 4.29 (1H, m); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 13.0, 14.1, 28.8, 29.4, 40.4, 42.1, 52.4, 70.3, 171.7, 175.2; ESI-TOFMS (pos.) m/z: 240.1200 [M + Na]⁺ (Calcd for C₁₀H₁₉NNaO₄: 240.1206).

General Synthesis Procedure for Compound B

To a solution of compound A (1.0 equiv.) in dichloromethane (0.3 mol/L) was added tetrabutylammonium hydrogensulfate (0.05 equiv.), AZADOL® (0.01 equiv.), and sodium hypochlorite pentahydrate (1.1 equiv.) at 0°C, followed by stirring at room temperature for one and a half hours. The reaction mixture was diluted with ethyl acetate and washed successively with a saturated solution of sodium bicarbonate in water, water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was chromatographed on a silica gel column to give compound B.

Methyl 2,5-Dioxo-5-(pyrrolidin-1-yl)pentanoate

Methyl 2-hydroxy-5-oxo-5-(pyrrolidin-1-yl)pentanoate (296 mg, 1.4 mmol) was used as the starting material. The chromatography on a silica gel column with n-hexane-ethyl acetate (1 : 9) gave methyl 2,5-dioxo-5-(pyrrolidin-1-yl)pentanoate (119 mg, 40% yield). ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.86 (2H, quin, J = 6.9), 1.97 (2H, quin, J = 6.9), 2.70 (2H, t, J = 6.3), 3.13 (2H, t, J = 6.5), 3.43 (2H, t, J = 6.1), 3.45 (2H, t, J = 6.7), 3.88 (3H, s); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 24.4, 26.0, 29.3, 33.7, 45.8, 46.5, 52.9, 161.3, 169.1, 193.3; ESI-TOFMS (pos.) m/z: 236.0889 [M + Na]⁺ (Calcd for C₁₀H₁₅NNaO₄: 236.0893).

Methyl 2,5-Dioxo-5-(piperidin-1-yl)pentanoate

Methyl 2-hydroxy-5-oxo-5-(piperidin-1-yl)pentanoate (240 mg, 1.1 mmol) was used as the starting material. The chromatography on a silica gel column with n-hexane-ethyl acetate (1 : 2) gave methyl 2,5-dioxo-5-(piperidin-1-yl)pentanoate (99 mg, 41% yield). ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.52 (2H, m), 1.60 (2H, m), 1.64 (2H, m), 2.76 (2H, t, J = 6.4), 3.10 (2H, t, J = 6.4), 3.42 (2H, t, J = 5.5), 3.52 (2H, t, J = 5.5), 3.88 (3H, s); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 24.4, 25.4, 26.3, 28.2, 33.8, 43.0, 46.5, 52.9, 161.3, 168.8, 193.3; ESI-TOFMS (pos.) m/z: 250.1047 [M + Na]⁺ (Calcd for C₁₁H₁₇NNaO₄: 250.1050).

Methyl 5-Diethylamino-2,5-dioxopentanoate

Methyl 5-(diethylamino)-2-hydroxy-5-oxopentanoate (1.1 g, 5.1 mmol) was used as the starting material. The chromatography on a silica gel column with hexane–ethyl acetate (1 : 2) gave methyl 5-(diethylamino)-2,5-dioxopentanoate (782 mg, 72% yield). ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.09 (3H, t, J = 7.1), 1.21 (3H, t, J = 7.1), 2.75 (2H, t, J = 6.2), 3.12 (2H, t, J = 6.2), 3.33 (2H, q, J = 7.1), 3.36 (2H, q, J = 7.1), 3.88 (3H, s); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 13.0, 14.1, 28.0, 34.1, 40.4, 41.9, 52.9, 161.3, 169.8, 193.3; ESI-TOFMS (pos.) m/z: 238.1054 [M + Na]⁺ (Calcd for C₁₀H₁₇NNaO₄: 238.1050).

2-Oxoglutaric Acid

Methyl 2-oxo-4-(N,N-diethylamido)butanoate (368 mg, 1.7 mmol) was added to 4.0 M hydrochloric acid (8.5 mL) and refluxed overnight. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in distilled water and filtered through a Dowex® 50WX8 column (Dupon, Wilmington, DE, U.S.A.). The filtrate was concentrated under reduced pressure to give 2-oxoglutaric acid (227 mg, 91% yield). ¹H-NMR (500 MHz, DMSO-d₆) δ ppm (J in Hz): 2.48 (2H, t, J = 6.5), 2.99 (2H, t, J = 6.5); ¹³C-NMR (125 MHz, DMSO-d₆) δ ppm: 27.4, 33.7, 162.2, 173.4, 195.0; ESI-TOFMS (neg.) m/z: 145.0142 [M − H]⁻ (Calcd for C₅H₅O₅: 145.0142).

Methyl [1-¹³C]5-Diethylamino-2-hydroxy-5-oxopentanoate

1) To a solution of N,N-diethyl-4-hydroxybutanamide (1.6 g, 10 mmol) in dichloromethane (30 mL) was added tetrabutylammonium hydrogensulfate (170 mg, 0.05 mmol), AZADOL® (15 mg, 0.1 mmol), and sodium hypochlorite pentahydrate (1.8 g,11 mmol) at 0°C, followed by stirring at room temperature for half an hour. The reaction mixture was diluted with ethyl acetate and washed successively with a saturated solution of sodium bicarbonate in water, water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude aldehyde was used for the subsequent reaction without further purification.

2) To a solution of the above aldehyde in dichloromethane (20 mL) was added TMS¹³CN (1.0 g, 10 mmol) and triethylamine (140 μL,1.0 mmol) at 0°C, followed by stirring at room temperature for one and a half hours. The reaction mixture was diluted with ethyl acetate and washed successively with water and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude cyanohydrin trimethylsilyl ether was used for the subsequent reaction without further purification.

3) To a solution of the above cyanohydrin trimethylsilyl ether in methanol (20 mL), chlorotrimethylsilane (6.3 mL, 50 mmol) was added and stirred at 50°C overnight. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in chloroform, and the precipitate was filtered off. The filtrate was concentrated under reduced pressure. The residue was chromatographed on a silica gel column with chloroform–ethyl acetate (19 : 1) to give methyl [1-¹³C]2-hydroxy-4-(N,N-diethylamido)butanoate (1.4 g, 3 steps, 65% yield). ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.13 (3H, t, J = 7.2), 1.18 (3H, t, J = 7.2), 2.01 (1H, m), 2.23 (1H, m), 2.50 (1H, m), 3.31 (2H, q, J = 7.2), 3.37 (2H, dq, J = 1.5, 7.2), 3.78 (3H, d, J_C–H = 3.7), 4.04 (1H, m, −OH), 4.28 (1H, m). ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 13.0, 14.1, 28.8, 29.4, 40.4, 42.1, 52.4 (d, J_C–C = 2.6), 70.3 (d, J_C–C = 60.7), 171.7, 175.2 (strong). ESI-TOFMS (pos.) m/z: 241.1241 [M + Na]⁺ (Calcd for C₉¹³CH₁₉NNaO₄: 241.1240).

Methyl [1-¹³C]5-Diethylamino-2,5-dioxopentanoate

To a solution of methyl [1-¹³C]2-hydroxy-4-(N,N-diethylamido)butanoate (1.3 g, 6.2 mmol) in dichloromethane (20 mL) was added tetrabutylammonium hydrogensulfate (100 mg, 0.3 mmol), AZADOL® (10 mg, 0.06 mmol), and sodium hypochlorite pentahydrate (1.1 g, 6.8 mmol) at 0°C, followed by stirring at room temperature for one and a half hours. The reaction mixture was diluted with ethyl acetate and washed successively with a saturated solution of sodium bicarbonate in water, water, and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was chromatographed on a silica gel column with hexane–ethyl acetate (1 : 2) to give methyl [1-¹³C]2-oxo-4-(N,N-diethylamido)butanoate (1.2 g, 93% yield) as a colorless oil. ¹H-NMR (500 MHz, CDCl₃) δ ppm (J in Hz): 1.09 (3H, t, J = 7.1), 1.21 (3H, t, J = 7.1), 2.75 (2H, t, J = 6.2), 3.12 (2H, t, J = 6.2), 3.33 (2H, q, J = 7.1), 3.36 (2H, q, J = 7.1), 3.88 (3H, d, J_C–H = 3.8); ¹³C-NMR (125 MHz, CDCl₃) δ ppm: 13.0, 14.1, 28.0, 34.1 (d, J_C–C = 16.0), 40.4, 41.9, 52.9 (d, J_C–C = 2.8), 161.3 (strong), 169.8, 193.3 (d, J_C–C = 69.0); ESI-TOFMS (pos.) m/z: 239.1083 [M + Na]⁺ (Calcd for C₉¹³CH₁₇NNaO₄: 239.1083).

¹³C-2OG

Methyl [1-¹³C]2-oxo-4-(N,N-diethylamido) butanoate (1.2 g, 5.6 mmol) was added to 4.0 M hydrochloric acid (25 mL) and refluxed overnight. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in distilled water and filtered through a Dowex® 50WX8 column. The filtrate was concentrated under reduced pressure to give ¹³C-2OG (728 mg, 89% yield). ¹H-NMR (500 MHz, DMSO-d₆) δ ppm (J in Hz): 2.48 (2H, t, J = 6.5), 2.99 (2H, t, J = 6.5); ¹³C-NMR (125 MHz, DMSO-d₆) δ ppm: 27.4, 33.7 (d, J_C–C = 15.3), 162.2 (strong), 173.4, 195.0 (d, J_C–C = 66.4); ESI-TOFMS (neg.) m/z: 146.0173 [M − H]⁻ (Calcd for C₅H₅O₅: 146.0176).

Animal Care

The Laboratory Animal Care Committee of Kawasaki University of Medical Welfare approved this study (No. 24-004). The rats were maintained in strict accordance with the Guidelines for the Care and Use of Laboratory Animals at Kawasaki University of Medical Welfare. Male Jcl:Wistar rats (CLEA Japan Inc., Tokyo, Japan) were housed individually in screen-bottomed, stainless steel cages. The cages were stored in a room maintained at 23 ± 1°C with a 12 h light/dark cycle. The rats were fed a diet (MF; Oriental Yeast Co., Ltd., Tokyo, Japan), and tap water was provided ad libitum for drinking.

¹³C Breath Test

The breath tests were performed according to the method of Uchida et al.¹⁷⁾ The rats were fasted for 17 h (overnight), and 24 rats weighing an average of 240 g at 8 weeks of age were allocated into 3 groups, each containing 8 rats. The rats were placed in a desiccator to acclimatize for about 30 min (approximately 6 L in capacity). Each rat was orally administered the ¹³C-labeled compound (120 μmol/kg) dissolved in saline (30 μmol/mL, Otsuka Normal Saline; Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan). The expired air was collected into bags (150–200 mL, Expired Air Collection Bag 20; Otsuka Pharmaceutical Co., Ltd.). The suction rate of exhaled air in the desiccator was 110 mL/min using a tube pump system (MasterFlex L/S, L/S24 tube; Cole-Parmer International, IL, U.S.A.). The time course of the increase in the ¹³CO₂/¹²CO₂ ratio (Δ¹³CO₂) in the air was measured by an IR analyzer (POCone; Otsuka Electronics Co., Ltd., Osaka, Japan). Commercially available standard gas (95% O₂/5%CO₂) was used as the reference CO₂ gas. The Δ¹³CO₂ values were used to construct the breath ¹³CO₂ expiration–time curve.

Statistical Analysis

All statistical analyses were performed using SPSS Statistics software, version 23 (IBM, Armonk, NY, U.S.A.). Data were analyzed by one-way ANOVA, and significant differences among the groups were determined using Tukey–Kramer’s test. Differences were considered significant at a p-value <0.05.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

References

• 1)  Arnold P. K., Finley L.W. S., J. Biol. Chem., 299, 102838 (2023).
• 2)  Suomalainen A., Nunnari J., Cell, 187, 2601–2627 (2024).
• 3)  Modac A. S., Chapter 17—¹³C breath tests. "Breathborne Biomarkers and the Human Volatilome (Second Edition)," Elsevier BV, Amsterdam, 2020, pp. 273–287.
• 4)  Wu I.-C., Ohsawa I., Fuku N., Tanaka M., Ann N. Y., Acad. Sci., 1201, 111–120 (2010).
• 5)  Karpusas M., Branchaud B., Remington S. J., Biochemistry, 29, 2213–2219 (1990).
• 6)  Lauble H., Kennedy M. C., Emptage M. H., Beinert H., Stout C. D., Proc. Natl. Acad. Sci. U.S.A., 93, 13699–13703 (1996).
• 7)  Mitome H., Takenishi M., Ono K., Kawagoe N., Imai T., Sasaki Y., Urita Y., Akira K., J. Labelled Comp. Radiopharm., 67, 86–90 (2024).
• 8)  Larsen B. D., Eggert H., Harrit N., Holm A., Spielbüchler P., Pedersen J. B., Krogsgaard-Larsen P., Acta Chem. Scand., 46, 482–486 (1992).
• 9)  Sasaki T., Takahashi S., Uchida K., Funayama S., Kainosho M., Nakagawa A., J. Antibiot., 59, 418–427 (2006).
• 10)  Madelaine C., Valerio V., Maulide N., Angew. Chem. Int. Ed., 49, 1583–1586 (2010).
• 11)  Meth-Cohn O., Taylor D. L., Tetrahedron, 51, 12869–12882 (1995).
• 12)  Huang P.-Q., Sheng X., Deng X.-M., Tetrahedron Lett., 42, 9039–9041 (2001).
• 13)  Kirihara M., Okada T., Sugiyama Y., Akiyoshi M., Matsunaga T., Kimura Y., Org. Process Res. Dev., 21, 1925–1937 (2017).
• 14)  Kobayashi S., Tsuchiya Y., Mukaiyama T., Chem. Lett., 20, 537–540 (1991).
• 15)  Recently, another synthetic method for ¹³C-2OG has been reported.: Miura N., Mushti C., Sail D., AbuSalim J. E., Yamamoto K., Brender J. R., Seki T., AbuSalim D. I., Matsumoto S., Camphausen K. A., Krishna M. C., Swenson R. E., Kesarwala A. H., NMR Biomed., 34, e4588 (2021). However, a major obstacle to scaling up this method is the requirement for stoichiometric amounts of the expensive 4-DMAP oxide to oxidize the alkyl halide into the corresponding aldehyde.
• 16)  Reez M. T., Chatziiosifidis I., Synthesis, 1982, 330 (1982).
• 17)  Uchida M., Endo N., Shimizu K., J. Pharmacol. Sci., 98, 388–395 (2005).