3 trimethylsilyl propionic 2 2 3 3 d4 acid sodium salt

The degree of functionalization was studied by using ¹H NMR according to a previously reported method [21 (link)]. ¹H NMR spectra were collected by using an NMR spectrometer (AL300; JEOL, Tokyo, Japan) with a single axis gradient inverse probe at a frequency of 300 MHz. Before the measurement, 30 mg of GelMA and GelMAGMA macromers were respectively dissolved into 1 mL deuterium oxide containing 0.05% (w/v) 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (Sigma-Aldrich, St. Louis, MO, USA) for calibration at 40 °C.

Sodium hydroxide, sodium chloride, aluminum chloride hexahydrate, p-toluensulfonic acid monohydrate (≥98.5), 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (95%), pyridine, chromium(iii)acetylacetonate, oleuropein, tyrosol, levulinic acid, 5-(hydroxymethyl)furfural, 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt, and pyrazine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric acid 37% was provided from Panreac (Barcelona, Spain). Ethyl acetate was obtained from Scharlau (Barcelona, Spain) while maleic acid, CDCl3 (+0.03% vol. TMS), deuterium oxide, and cyclohexanol were purchased from Fluka™ (St. Gallen, Switzerland), VWR Chemicals (Solon, OH, USA), and Acros Organics (Geel, Belgium), respectively.

D-Glucose ( $\ge$  99.5%), 13-C labelled D-Glucose ( $\ge$  99.5%), 3-(trimethylsilyl)- propionic-2,2,3,3- ${\text{d}}_4$ acid sodium salt (99.9 atom %, TSP), deuterium oxide ${\text{D}}_2\text{O}$ (99.9 atom % D), phosphate buffer saline (PBS) tablets, boric acid (ACS reagent, $\ge$  99.5%), and sodium dihydrogen phosphate (BioReagent, for molecular biology, anhydrous, $\ge$  98%) were purchased from Sigma-Aldrich without further purification.
The acqueous test sample was prepared as 500 mM 13-C labelled D-Glucose, 500 mM boric acid, 500 mM ${\text{NaH}}_2{\text{PO}}_4$ and 250 mM TSP dissolved in 3 mL of a 500 mM PBS solution (pH 5.5, using PBS tablets) prepared using 50% ${\text{H}}_2\text{O}$ and 50% ${\text{D}}_2\text{O}$ .

The degree of methacryloyl functionalization was quantified by using ¹H NMR according to a previously described method [36 (link)]. ¹H NMR spectra were collected by using a NMR spectrometer (JEOL Co. Ltd., Tokyo, Japan) with a single axis gradient inverse probe at a frequency of 300 MHz. Before the measurement, 20 mg of GelMA macromers was completely dissolved in 1 mL deuterium oxide containing 0.05 wt % 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid sodium salt for calibration (Sigma-Aldrich, St. Louis, MO, USA). The gelatin without functionalization was also examined for calculating the degree of methacryloyl substitution using the following Equation (1):

The purified 5-hydroxyhomoectoine was employed to elucidate the stereochemical configuration of hydroxyhomoectoine, in particular to reveal the carbon atom at which the hydroxylation takes place and with which selectivity. Broad-band-decoupled ¹H NMR spectra and 2d-NMR spectra were recorded at 300 K on a Bruker AV III HD 500 MHz spectrometer in deuterium oxide (99.9% D) containing 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (internal standard) (Sigma-Aldrich Chemie GmbH, München, Germany) at 500.13 MHz or on a Bruker AV II 600 MHz in H₂O using solvent suppression excitation sculpting with gradients at 600.13 MHz. ¹³C NMR spectra were recorded on a Bruker AV II 300 at 75.49 MHz. Chemical shifts are reported in ppm relative to the solvent-residue signal (¹H spectra) or to the internal standard (¹³C spectra), respectively. Multiplicities are given as singlet (s), doublet (d), triplet (t), quartet (q), quintet (quin), multiplet (m), and broad (b) where applicable. Spectra were processed with Bruker TopSpin^® software 4.0.

All chemicals and reagents used were of analytical grade. Potassium dihydrogen phosphate (99%, KH₂PO₄), deuterium oxide (99.9%, D₂O), methanol-d₄ (>99.8%, MeOD), and methanol were purchased from VWR (Radnor, PA, United States). Sodium deuteroxide 40% w/v solution in D₂O (99.5%, NaOD) was obtained from Alfa Aesar (Kandel, Germany). 3-(Trimethylsilyl) propionic-2,2,3,3-d₄ acid sodium salt (99%, TMSP) was obtained from Sigma-Aldrich (St. Louis, MO, United States). A Millipore Direct-Q^® 3 UV Water Purification System (Millipore Corp., Bedford, MA, United States) or (Merck KGaA, Darmstadt, Germany) for ultrapure water was used throughout the study.