A Versatile Broadband Attached Proton Test Experiment for Routine 13C Nuclear Magnetic Resonance Spectroscopy

Abstract

The proposed broadband attached proton test sequence allows the user to easily record ¹³C nuclear magnetic resonance multiplicity-edited and quaternary-carbon-only spectra. Compared to earlier attached proton test experiments, it preserves both a tolerance for wide ranges of one-bond-coupling constant values and the effective suppression of residual CHn signals in the quaternary-carbon-only spectra. The recording of edited spectra or quaternary-carbon-only spectra is made easy by a single, user-controllable constant. These attributes make the broadband attached proton test experiment attractive for the ¹³C analysis of small molecules, including spectral editing, particularly in high-throughput analysis laboratories.

Keywords: 13C; 1H; 1JCH-tolerance; APT; Cq-only; NMR; broadband APT.

Figure 1

Pulse sequence of the CAPT3 (APTjc in the Bruker pulse program library, top) and the proposed broadband APT (BAPT, bottom) experiments. Thin bars stand for 90° pulses, thick bars for 180° pulses. All ¹³C 180° pulses can be replaced by broadband refocusing pulses [34,35]. The first ¹³C pulse may be set shorter than 90° to allow for a faster repetition rate (shown in grey). In the APTjc experiment, Δ₁ is set to an average value 1/(2¹J¹_CH) [24]. In the BAPT experiment, Δ₁, Δ₂, and Δ₃ are set as described in the text, with Δ₂ = 1/J²_CH to record APT-like spectra and Δ₂ = 1/(2*J²_CH) to record Cq-only spectra. The following phase cycling is applied: φ₁ = 4(x), 4(−x), 4(y), 4(−y), φ₂ = x, y, −x, −y, y, −x, −y, x, −x, −y, x, y, −y, x, y, −x, φ₃ = x, y, y, x,( y, x, x, y)₂, x, y, y, x, φ_REC = x, x, −x, −x, y, y, −y, −y. BAPT experiment: φ₄ = x (APT-like mode); x, x, −x, −x (Cq-only mode), φ₅ = −x (APT mode); y, −y (Cq-only mode). Phases not shown are applied along the x-axis.

Figure 2

Theoretical intensity of CH, CH₂, and CH₃ groups as a function of the ¹J_CH value for the APT pulse sequence, Δ₁ = 1/(¹J¹_CH) (--), the APTjc pulse sequence, Δ₁ = 1/(2*¹J¹_CH) (--), and the BAPT pulse sequence, Δ₁ = 1/(2*¹J¹_CH), Δ₂ = 1/¹J²_CH, and Δ₃ = 1/(2*¹J³_CH) (--). For APT and APTjc, Δ₁ was set to match a coupling constant value of 145 Hz. For BAPT, Δ₁ and Δ₃ were set to match a coupling constant value of 130 Hz, and Δ₂ was set to match a coupling constant value of 175 Hz. Three quaternary carbons at 35, 45, and 55 ppm have been added for comparison and are shown with ●. For CH groups, the ¹J_CH value was varied from 135 to 175 Hz (right to left) in steps of 5 Hz. For CH₂ groups, the ¹J_CH value was varied from 125 to 165 Hz (right to left) in steps of 5 Hz. For CH₃ groups, the ¹J_CH value was varied from 110 to 130 Hz (right to left) in steps of 2.5 Hz. Full relaxation was considered. Simulations were performed using NMRSIM for Windows.

Figure 3

Theoretical intensity of CH, CH₂, and CH₃ groups as a function of the ¹J_CH value for the APT pulse sequence, Δ₁ = 1/(¹J¹_CH) (--), the APTjc pulse sequence, Δ₁ = 1/(2*¹J¹_CH) (--), and the BAPT pulse sequence, Δ₁ = 1/(2*¹J¹_CH), Δ₂ = 1/¹J²_CH, and Δ₃ = 1/(2*¹J³_CH) (--). For APT and APTjc, Δ₁ was set to match a coupling constant value of 145 Hz. For BAPT, Δ₁ and Δ₃ were set to match a coupling constant value of 145 Hz, and Δ₂ was set to match a coupling constant value of 250 Hz. Three quaternary carbons at 35, 45, and 55 ppm have been added for comparison and are shown with ●. For CH groups, the ¹J_CH value was varied from 135 to 250 Hz (right to left) in steps of 15 Hz. For CH₂ groups, the ¹J_CH value was varied from 125 to 165 Hz (right to left) in steps of 5 Hz. For CH₃ groups, the ¹J_CH value was varied from 110 to 130 Hz (right to left) in steps of 2.5 Hz. Full relaxation was considered. Simulations were performed using NMRSIM for Windows.

Figure 4

Theoretical residual intensity of CH, CH₂, and CH₃ groups in a Cq-only spectrum as a function of the ¹J_CH value for the APT pulse sequence, Δ₁ = 1/(2*¹J¹_CH) (--), the modified APTjc pulse sequence, Δ₁ = 1/(2*¹J¹_CH) (--), and the BAPT pulse sequence, Δ₁ = 1/(2*¹J¹_CH), Δ₂ = 1/(2*¹J²_CH), and Δ₃ = 1/(2*¹J³_CH) (--). The spin system and all other parameters were identical to those used for Figure 2.

Figure 5

Theoretical residual intensity of CH, CH₂, and CH₃ groups in a Cq-only spectrum as a function of the ¹J_CH value for the APT pulse sequence (--), the APTjc pulse sequence, Δ₁ = 1/(2*¹J¹_CH) (--), and the BAPT pulse sequence, Δ₁ = 1/(2*¹J¹_CH), Δ₂ = 1/(2*¹J²_CH), and Δ₃ = 1/(2*¹J³_CH) (--). The spin system and all other parameters were identical to those used for Figure 3.

Figure 6

APT (A), APTjc (B), and BAPT (C) spectra of cholesteryl acetate. For APT and APTjc, the delay, Δ, was adjusted to a coupling constant of 145 Hz, while for BAPT, Δ₁ and Δ₃ were adjusted to a coupling constant of 130 Hz, and Δ₂ was adjusted to a coupling constant of 170 Hz.

Figure 7

Molecular structure of cholesteryl acetate and atom numbering used in the text.

Figure 8

Resonances of C12 (δ = 12 ppm, ¹J_CH = 122 Hz) of cholesteryl acetate and of TMS (δ = 0 ppm, ¹J_CH = 117 Hz) dissolved in 0.7 mL of CDCl₃ recorded with the APT (black), APTjc (red), and BAPT (green) experiments.

Figure 9

Cq-only spectra of cholesteryl acetate obtained using the APT (A), modified APTjc (B), and BAPT (C) pulse sequences. For APT and APTjc, the delay Δ was adjusted to a coupling constant of 145 Hz, while for BAPT, Δ₁ and Δ₃ were adjusted to a coupling constant of 130 Hz, and Δ₂ was adjusted to a coupling constant of 170 Hz.

Figure 10

Molecular structure of 4-methyl-N,N-(prop-2-yn-1-yl)aniline and atom numbering used in the text.

Figure 11

APT (A), APTjc (B), and BAPT (C) spectra of ~10 mg of 4-methyl-N,N-di(prop-2-yn-1-yl)aniline dissolved in 0.7 mL of CDCl₃. For APT and APTjc, the delay Δ₁ was adjusted for a coupling constant ¹J¹_CH of 145 Hz. For BAPT, the delays Δ₁ and Δ₃ were adjusted for a coupling constant ¹J^1,3_CH of 145 Hz, and the delay Δ₂ was adjusted for a coupling constant ¹J²_CH of 240 Hz. The relaxation delay was 2 s. All other parameters were identical to those described in the Materials and Methods Section.

Figure 12

Resonances of C1, C3, C5, C6, and C8 of ~10 mg of 4-methyl-N,N-di(prop-2-yn-1-yl)aniline dissolved in 0.7 mL of CDCl₃ recorded with the APT (green), APTjc (red), and BAPT (black) experiments.

Figure 13

APT (A), APTjc (B), BAPT (C), and SEMUT-GL (D) Cq-only spectra of ~10 mg of 4-methyl-N,N-di(prop-2-yn-1-yl)aniline dissolved in 0.7 mL of CDCl₃. For the APT and the modified APTjc experiments, the delay Δ₁ was adjusted for a coupling constant ¹J¹_CH of 145 Hz. For BAPT, the delays Δ₁ and Δ₃ were adjusted for a coupling constant ¹J^1,3_CH of 145 Hz, and the delay Δ₂ was adjusted for a coupling constant ¹J²_CH of 240 Hz. For SEMUT-GL, the delay Δ₁ was adjusted for a coupling constant ¹J^1,3_CH of 133 Hz, the delay Δ₂ was adjusted for a coupling constant ¹J²_CH of 235 Hz, and the delay Δ₃ was adjusted for a coupling constant ¹J^1,3_CH of 191 Hz [27,32]. The tip angle of the first ¹³C pulse was 30°, and the relaxation delay was 4 s. All other parameters were identical to those described in the Materials and Methods Section.

Figure 14

Molecular structures of oleic acid, linoleic acid, and the diruthenium–coumarin conjugate 16a [36], and atom numbering used in the text.

Figure 15

(Top): part of the 2D ¹H-¹³C edited HSQC spectrum of an equimolar (30 mmol) mixture of oleic acid, linoleic acid and compound 16a dissolved in CDCl₃. CH and CH₃ signals are phased up (black peaks) and CH₂ signals are phased down (red peaks). (Bottom): BAPT spectrum of the same mixture, CH and CH₃ signals are phased up and CH₂ signals are phased down. The experimental parameters of the BAPT experiment were identical to those used for Figure 6.

Figure 16

Part of the 7 Hz adjusted HMBC spectrum of cholesteryl acetate. Top: BAPT CHn, Cq spectrum set as external F₁ projection. Bottom: BAPT Cq-only spectrum set as external F₁ projection. The experimental parameters of the BAPT experiments were identical to those used for Figure 9.