Chemoselective detection and discrimination of carbonyl-containing compounds in metabolite mixtures by 1H-detected 15N nuclear magnetic resonance

Abstract

NMR spectra of mixtures of metabolites extracted from cells or tissues are extremely complex, reflecting the large number of compounds that are present over a wide range of concentrations. Although multidimensional NMR can greatly improve resolution as well as improve reliability of compound assignments, lower abundance metabolites often remain hidden. We have developed a carbonyl-selective aminooxy probe that specifically reacts with free keto and aldehyde functions, but not carboxylates. By incorporating (15)N in the aminooxy functional group, (15)N-edited NMR was used to select exclusively those metabolites that contain a free carbonyl function while all other metabolites are rejected. Here, we demonstrate that the chemical shifts of the aminooxy adducts of ketones and aldehydes are very different, which can be used to discriminate between aldoses and ketoses, for example. Utilizing the 2-bond or 3-bond (15)N-(1)H couplings, the (15)N-edited NMR analysis was optimized first with authentic standards and then applied to an extract of the lung adenocarcinoma cell line A549. More than 30 carbonyl-containing compounds at NMR-detectable levels, six of which we have assigned by reference to our database. As the aminooxy probe contains a permanently charged quaternary ammonium group, the adducts are also optimized for detection by mass spectrometry. Thus, this sample preparation technique provides a better link between the two structural determination tools, thereby paving the way to faster and more reliable identification of both known and unknown metabolites directly in crude biological extracts.

Keywords: 15N-edited 1H NMR; aldehydes; aminooxy derivatives; chemoselection; ketones; metabolomics.

Figure 1. Structure of the ¹⁵N-QDA and adducts

A: ¹⁵N- QDA reagent B: reaction of ¹⁵N-QDA with aldehydes and ketones and long range ¹H-¹⁵N scalar couplings in the oxime adducts.

Figure 2

NMR ¹⁵N-edited NMR spectra of ¹⁵N-QDA adducts of standards and A549 cell extract. The samples were prepared as described in the Experimental Procedures. 1D NMR spectra were recorded at 18.8 T. The 1D ¹H-{¹⁵N} HSQC spectrum was recorded using an INEPT delay of 3 ms with an acquisition time of 0.5 s and recycle time of 2 s. The 1D unedited spectrum was acquired similarly with an acquisition time of 2 s and a recycle time of 5 s. Total acquisition times were 34 min. Top : 1D HSQC Spectrum of standards Middle 1D HSQC Spectrum of the polar extract of A549 cells Bottom : 1D presat spectrum of the A549 cell extract.

Figure 3. Long range ¹H{¹⁵N} HSQC of a mixture of standard ¹⁵N-QDA adducts

The mixture of 17 standards was dissolved in phosphate buffer. The ¹⁵N HSQC spectrum was recorded at 20 °C 18.8 T with acquisition times of 0.15 s in t₂ and 0.016 s in t₁. The data tables were zero filled once in each dimension and apodised using an unshifted Gaussian function and 1 Hz line broadening exponential. The J_NH INEPT delay was set to 83.3 ms. ¹⁵N chemical shifts were referenced indirectly with respect to external liquid ammonia ^[20]. Red and cyan boxes shows resonances from aldehydes and ketones, respectively.

Figure 4. Correlation of ¹H and ¹⁵N chemical shifts for ¹⁵N-QDA adducts of standards

Chemical shifts of the ¹⁵N-QDA adducts were determined at 20 °C as described in the Experimental Procedures. ¹⁵N chemical shifts were referenced indirectly with respect to external liquid ammonia ^[20]. squares : ketones, circles : aldehydes δ_N= 325.1±1.59+5.94±0.26 δ_H r²=0.975 (n=16). ²J_NH (aldehydes) = 2.11±0.2 Hz ³J_NH (ketones) = 2.64±1.1 Hz Ketones and aldehydes are readily distinguished based on their ¹H and ¹⁵N chemical shifts.

Figure 5. ¹H{¹⁵N} HSQC spectra of QDA* adducts of an extract of A549 cells

The A549 polar extract was prepared and derivatized as described in the Experimental Procedures. The dried sample was redissolved in 100% CD₃OD. HSQC spectra were recorded at 14.1 T with acquisition times of 0.5 s in t₂ and 25.6 ms in t₁. The data tables were zero filled once in t₂, linear predicted once in t₁ and zero filled to 8192×2048 points. The free induction decays were processed using an unshifted Gaussian and a line broadening exponential of 4 Hz in both dimensions. ¹⁵N chemical shifts were referenced indirectly with respect to external liquid ammonia ^[20]. Some assignments are shown on the figure.