*4.5. NMR Spectroscopy*

Mono- and two-dimensional NMR experiments were recorded on a 500-MHz Bruker DMX spectrometer, operating at 11.7 T, equipped with a 5 mm probe and gradient unit on z, and thermostated at 298 K (Bruker Biospin GmbH, Rheinstetten, Karlsruhe, Germany). The samples were prepared by dissolving 5.0 mg of oxidized starch into 700 μL of D2O at room temperature (The NMR data were acquired on modified PS before the lyophilization process, and no precipitate or solid materials was observed in the NMR tube.). As internal standard, 20 μL of a 0.7 mM TMS/water solution was added to each NMR solution before data acquisition.

Acquisition parameters for 1H experiments of enzymatically oxidized pea starch: 90◦ pulse 9.75 μs; PL1−2.2 dB; relaxation delay, 20.0 s. Spectral width, 8400 Hz; number of transient, 1024.

13C parameters: spectral width, 14 KHz, 90◦ pulse, 11.0 μs; PL1−1.3 dB with a delay of 10 s.

2D 1H-1H DQF-TOCSY (double quantum filtered-total correlation spectroscopy) was acquired with 256 experiments over 2 K data points and 256 scans each, with a mixing time of 0.09 s and a relaxation delay of 1.2 s.

The 2D 1H-13C g-HSQC experiments (gradient-heteronuclear single quantum coherence) were performed by applying a coupling constant *1JCH* = 150 Hz; data matrix 2 K × 256; number of scans: 128.

The 2D 1H-13C g-HMBC experiments (gradient-heteronuclear multiple bond correlation) were performed by applying a delay of 50 ms for the evolution of long-range coupling; data matrix 2 K × 256; number of scans 128; D1 2.00 s. Data were zero filled and weighted with a sine bell function before Fourier transformation.

Quantitative acquisition parameters for 1H spectra of ca ffeine solutions: 90◦ pulse, 9.75 μs; PL1−2.2 dB; relaxation delay, 40.0 s. Spectral width, 8400 Hz; number of transient, 1024. Data processing: exponential line broadening of 0.1 Hz was applied as resolution enhancement function; zero-filling to 32 K prior FT (TopSpin 4.0.6 software, Bruker Biospin GmbH, Rheinstetten, Karlsruhe, Germany). Spectra were referenced to the residual solvent signal of TMS at δ = 0 ppm, as internal standard.

For all experiments, spectra phasing and integration were performed manually, and the NMR spectra were processed using the Bruker TopSpin 4.0.6 software (Bruker Biospin GmbH, Rheinstetten, Karlsruhe, Germany).

Ca ffeine content was determined from the integral value in the proton spectrum by applying Equation (1) [36]:

$$\text{[mM]c = lc/Flc [mM]st Flst/lst} \tag{1}$$

where [mM]c is the millimolar concentration of ca ffeine; [mM]st is the millimolar concentration of the standard solution of TMS; Ic, Ist, and Hc and Hst are the integral value and number of protons generating the signals of ca ffeine and TMS, respectively. The 1H spectrum of ca ffeine with resonance assignment is reported in Figure S2.
