Ultra-high-resolved HSQC spectra of multiple- 13C-labeled biofluids.

The NMR analysis of cell extracts and culture media from multiplied by an exponential weighting function, giving an additional line broadening of 2 Hz. cells fed with multiple-C-labeled substrates such as [UC6]glucose gives access to various aspects of cellular meF98 glioma cells were grown to confluency in 15 cm culture dishes in a humidified atmosphere of 10% CO2 in tabolism. C NMR allows the simultaneous identification of individual C isotopomers in order to distinguish different air at 377C in DMEM, supplemented with 5% FCS and penicillin/streptomycin (100 units /ml) . For cell extracts, metabolic pathways. These measurements are commonly carried out with 1D carbon spectroscopy (1–6) . The advanapproximately 10 cells obtained from four culture dishes were incubated for 24 h in DMEM medium containing 8 mM tage of this method is that all labeled carbons are detected in one spectrum with very high spectral resolution. The dis[U-C6]glucose. All samples were adjusted to pH values of 7–8. advantages, however, are the low sensitivity, the signal superpositions, and the need to assign unknown resonances Sensitivity-improved HSQC with gradient echo/antiecho selection is used for optimum sensitivity (10, 11) . There are based on chemical shifts only. In contrast, 2D inverse H,C spectroscopy offers better signal separation, very high sensiseveral possibilities for introducing a selective pulse into this pulse sequence. The sequence in Fig. 1a replaces the tivity, and tools to confirm the assignment (7) . A drawback is the low resolution in the carbon dimension. Especially at carbon 907 excitation pulse with a G4 Gaussian pulse cascade (12) , and the sequence in Fig. 1b replaces the 1807 high field (600/800 MHz), it is almost impossible to resolve C–C couplings with an acceptable number of t1 increments pulse in the first INEPT step with a Gaussian 1807 pulse. The pulse sequence in Fig. 1a is similar to the pulse sequence in a nonselective HSQC experiment. To overcome this limitation, we have recently proposed a region-selective HSQC presented in (8) , but is now combined with sensitivity improvement. The advantage of the new pulse sequence shown (8) . Here we present an improved version and optimum combination with J scaling and constant-time evolution. Apin Fig. 1b is that it is of the same length as the nonselective HSQC with a signal intensity of approximately 120% complications to a multilabeled cell extract of F98 glioma cells are shown. pared to sequence 1a. However, the advantage of sequence 1a is an almost rectangular excitation profile of the G4 All experiments were performed on a Bruker DRX 600 MHz spectrometer. A 5 mm, H,C,N, inverse triple-resonance Gaussian pulse cascade. Sequence 1b requires a symmetrical pulse. We used a Gaussian 1807 pulse, but other symmetrical probe with shielded gradients was used. Gradients were shaped by a waveform generator and amplified by a Bruker pulses like hyperbolic secant are also possible. A useful feature of 2D spectroscopy is J scaling (13) . Acustar amplifier. Sinusoidal z gradients of duration 1 ms and recovery time 100 ms have been used for echo/antiecho The evolution of the chemical shift can be manipulated by inserting 1807 pulses into the evolution time t1 . In our case, gradient selection. Gradient fine adjustment (40:10.08) has been performed to get optimum intensity. Low-power adiaan upscaling of the C– C coupling constants was required. This can be obtained by introducing an additional 1807 carbatic composite-pulse decoupling with WURST (9) has been used. A Gaussian 1807 pulse of length 1 ms was used to bon pulse and adding several incremented d0 delays (Fig. 1c) . JCC evolution occurs during the full t1 period, whereas excite a range of 15 ppm in F1( C). The selective HSQC experiments were acquired with 512 or 1024 t1 increments, C chemical shift evolves only during the last two d0 delays. This version scales up the C– C coupling by a factor of for a spectral width of 15 ppm, to give a digital resolution in the carbon dimension of 4 or 2 Hz/pt. An acquisition 3. Additional d0 delays cause an even greater upscaling but may cause signal loss due to relaxation. J scaling offers time of 285 ms has been used to acquire a spectral width of 3 ppm in the proton dimension using digital quadrature the opportunity to unravel superimposed lines by utilizing variable line splitting. This holds, for example, if one eledetection. For the carbon spectra, an H,C dual probe was used with acquisition time 0.9 s and repetition time 2 s. This ment of a multiplet pattern is superimposed on a singlet signal of a monolabeled C isotopomer. affords a digital resolution of 0.55 Hz/pt. The spectra were