NMR Analysis of Carboxylate Isotopomers of 13C-Metabolites by Chemoselective Derivatization with 15N-Cholamine

Abstract

A substantial fraction of common metabolites contains carboxyl functional groups. Their ¹³C isotopomer analysis by nuclear magnetic resonance (NMR) is hampered by the low sensitivity of the ¹³C nucleus, the slow longitudinal relaxation for the lack of an attached proton, and the relatively low chemical shift dispersion of carboxylates. Chemoselective (CS) derivatization is a means of tagging compounds in a complex mixture via a specific functional group. ¹⁵N₁-cholamine has been shown to be a useful CS agent for carboxylates, producing a peptide bond that can be detected via ¹⁵N-attached H with high sensitivity in heteronuclear single quantum coherence experiments. Here, we report an improved method of derivatization and show how ¹³C-enrichment at the carboxylate and/or the adjacent carbon can be determined via one- and two-bond coupling of the carbons adjacent to the cholamine ¹⁵N atom in the derivatives. We have applied this method for the determination of ¹³C isotopomer distribution in the extracts of A549 cell culture and liver tissue from a patient-derived xenograft mouse.

Figure 1:. Time course of ¹⁵N₁-cholamine tagging reaction in a mixture of amino acids.

A mixture of Ala, Val, Gly, Phe (1 mM each compound) was reacted with ¹⁵N-cholamine in ratios of 1: 15: 60 at 60 °C in H₂O. The formation of the peptide derivatives was monitored at three time points (0 h, 1 h and 2 h). (A) 1D ¹H-NMR spectra (δ 10 – 0 ppm) were recorded at 16.45 T and processed as described in the Experimental Section. (B, C) Expansion of A, representing the aromatic/peptide region (B) and methyl regions (C), where the unmodified amino acids were marked with dashed boxes. D) Changes in the peak intensities of the unmodified amino acids during the reaction with ¹⁵N-cholamine with respect to the original

Figure 2.. 2D ¹H{¹⁵N}-HSQC spectrum of ¹⁵N₁-cholamine derivatives of 29 standards mixture.

The standard mixture was reacted for 2 h at 60° with ¹⁵N₁-cholamine and DMTMM in ratios of 1: 15: 60 in H₂O as described in the Experimental Section. The spectrum was recorded at 16.45 T and processed as described in the Experimental Section. Assignment of carboxyl-containing metabolites mixture tagged with ¹⁵N₁-cholamine: 1. Leu, 2. Ile, 3. Val, 4. Ala, 5. Glu, 6. Gln, 7. Asp, 8. Gly, 9. Phe, 10. His, 11. Tyr, 12. Trp, 13. Ser, 14. Thr, 15. Cys, 16. Cystine, 17. N-acetyl-Asp, 18. Lactate, 19. Pyruvate, 20. Formate, 21. Acetate, 22. Fumarate, 23. Citrate, 24. Malate, 25. Succinate, 26. α-KG, 27. 3-phosphoglycerate, 28. GSH, 29.

Figure 3.. Simulated and observed cross-peak splitting patterns in 2D ¹H{¹⁵N}-HSQC spectra of different mixtures of acetate isotopomers.

A) Structure of the product of acetate reacted with ¹⁵N-cholamine showing the different coupling constants between ¹³C-¹⁵N and ¹H_N-¹H. B-I) Side by side comparison of the simulated and observed peak patterns of different acetate isotopomer mixtures. The following peak patterns are evident in the ¹⁵N dimension: ¹²CH₃¹²CO₂- a singlet (B), ¹²CH₃¹³CO₂- a doublet with ¹J_CN = 14.4 Hz (C), ¹³CH₃¹²CO₂- a doublet with ²J_CN = 8 Hz (D), ¹²CH₃¹³CO₂- + ¹³CH₃¹²CO₂- two overlapping doublets or an apparent triplet (E), ¹²CH₃¹³CO₂- + ¹³CH₃¹²CO₂- + ¹²CH₃¹²CO₂- an apparent triplet plus singlet (F), ¹³CH₃¹³CO₂- a doublet of doublet (G); ¹²CH₃¹²CO₂- + ¹³CH₃¹³CO₂- a doublet of doublet plus overlapping singlet (H), and ¹²CH₃¹³CO₂- + ¹³CH₃¹³CO₂- a doublet of doublet plus a doublet (I). Different colors indicate different ¹³C label positions in the acetate isotopomers: Gray, ¹²C; Yellow, ¹³CO; Blue, ¹³CA; Purple, ¹³CO-¹³CA; Green, overlapping of yellow and blue peaks. Pixels are scaled to the coupling constants to accurately represent the splitting patterns. The chemical shift scales in both dimensions are shown in panel H, which is the same for all panels.

Figure 4. Analysis of ¹⁵N₁-cholamine tagged carboxyl groups in human lung adenocarcinoma A549 cells and mouse PDX liver tissue extracts.

[¹³C₆]-glucose treatment, polar extraction, and NMR experiment setups were as described in the Experimental Section. A) High resolution 2D ¹H{¹⁵N}-HSQC spectrum of ¹⁵N₁-cholamine derivatized extract of A549 cells cultured in [¹³C₆]-glucose enriched DMEM media. Peak assignments are shown in Table S1. Ala (4), lactate (18), Glu (5), and Gly (8) peaks showed signature splitting patterns in the N dimension, which confirms ¹³C labeling at the carboxylate (C1) and/or the neighboring carbon (C2) in these metabolites. B) High resolution 2D ¹H{¹⁵N}-HSQC spectrum of ¹⁵N₁-cholamine derivatized extract of PDX mouse liver tissue after liquid diet administration of ¹³C₆-glucose. Lactate, alanine, glutamate, glutamine, GSH/GSSG showed ¹³C enrichment in the N dimension. C) The first increment of HNCO experiment in the HCO plane of the PDX mouse liver tissue extract reacted with ¹⁵N₁-cholamine. It confirmed ¹³C labeling at the carboxylate group of these metabolites . D) The first increment of HNCO experiment in the HN plane of the PDX mouse liver tissue extract reacted with ¹⁵N₁-cholamine, confirming ¹⁵N labeling in these metabolites.