In situ characterization of advanced glycation end products (AGEs) in collagen and model extracellular matrix by solid state NMR

Pathological glycation of extracellular matrix modelled with 13C-labelled sugars yields unique novel atomic level NMR structural and chemical insights non-destructively.


Materials and Methods
U-13 C 5 ribose was purchased from Cambridge Isotope Laboratories (Andover, MS USA) and bovine Achilles tendon type I collagen was from Sigma.
VSMCs were harvested from fresh adult bovine aortae. Cell culture reagents, media, suppliers, harvesting of VSMCs from bovine aortae, and their use in preparation of ECM, basically follows the methods used for fetal sheep osteoblast ECM described in detail in the supplementary material for Chow et al 1 . VSMCs were released from aortic strips by collagenase treatment and cultured to confluence in basal minimum Eagle's (BME) complete medium. Incubation was at 37°C in humidified 95% air, 5% CO2. Media were renewed every 2 days until the cells and matrix began to detach from the culture flask, generally after ca. 9 days, by which time enough ECM had formed for SSNMR. Cells were washed with phosphate buffered saline (PBS). After 24 hours at -80ºC the cells were freeze-thaw lysed, debris removed by repeated PBS washes, and decellularized ECM dislodged by swirling the flask in PBS, centrifuged at (1200 rpm, 5 min, room temperature) and stored at -20°C until incubation with D-(U-13 C 5 ) -R5P.
Dry collagen was rehydrated overnight in aqueous acetic acid (10%), plunger homogenized, and washed with distilled water and centrifuged repetitively until no longer acidic, and stored wet at 4 °C until used. Bovine collagen, and bovine VSMC ECM, were incubated in 6 mL of (U-13 C 5 )-ribose-5-phosphate (50 mM; see next section for synthesis) in 50mM sodium phosphate buffer at pH 7.4 and 37 ºC for 11 weeks, comparably to incubations with U-13 C 5 -Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2017 ribose reported in Bullock et al 2 .. Solution-state 13 C NMR spectra were recorded on the supernatant periodically to monitor glycation progress by the generation of small soluble glycation products. After incubation both glycated collagen, and glycated ECM, were washed several times with deionised water to remove unreacted sugar, phosphate, and soluble glycation products, freeze dried, and packed into a Bruker 4 mm o.d. zirconia MAS rotor (glycated collagen), or a Kel-F disposable insert (VSMC ECM) and stored at -20 ºC until ssNMR analysis. Scheme. a) Hydrochloric acid, acetone/methanol (1:

D-(U-13 C 5 )-ribofuranoside-5-phosphate disodium salt (4)
Compound 3 (430 mg, 1.51 mmol) was dissolved in water (1 mL) and trifluoroacetic acid was added to pH = 1. The solution was stirred at rt for 2 h and concentrated under diminished pressure, then the crude was purified through a Sep-Pak C 18 cartridge. After the purification, the compound was eluted with water on an ion-exchange column of Dowex ® 50WX8 (Na +

Solid state NMR
All experiments were carried out on Bruker AVANCE II 400 MHz wide bore spectrometers using 4 mm rotors. All extracellular matrix samples were packed into disposable inserts (Bruker) designed to be used inside these rotors. Cross-polarisation (CP) -magic-anglespinning (MAS): Typical parameters: MAS frequency 10 kHz, 1 H 90° pulse 2.5 μs, contact time of 2.5 ms with ramped pulse on 1 H and square pulse on 13 C or 15 N at 70 kHz spin lock field strength, 70 kHz field strength SPINAL64 decoupling during acquisition, recycle delay 2s.
Single quantum-double quantum 13 C-13 C correlation experiments (SQ-DQ): Initial CP parameters as for 13 C CP experiments. At 10 kHz MAS, a 70 kHz POST-C7 pulse sequence 3 was applied on 13 C channel to excite double quantum coherence in 0.4 ms. Magnetisation was returned to zero quantum by another 0.4 ms of POST-C7 sequence. During double quantum evolution, 100 kHz Lee-Goldberg decoupling was applied on 1 H.
Proton-driven spin diffusion 13 C-13 C correlation experiments (PDSD): Initial CP parameters as for 13 C CP experiments. The magnetisation was allowed to evolve at single-quantum coherence during the incremental delay, and returned to zero quantum coherence by a 13 C 90° pulse (3.57 μs). 1 H decoupling was switched off during this mixing period ( M 20 ms and 100 ms in separate experiments), with a 13 C 90° readout pulse at the end of the mixing period. During both the incremented delay and acquisition periods, SPINAL64 decoupling was applied on 1 H at 70 kHz.