*2.3. NMR Analysis*

The Ch and HA spectra were obtained with a Bruker AVANCE IIIHD 500 MHz spectrometer (Bruker, Karlsruhe, Germany) equipped with a 5 mm BBO probe, at 343 K. Spectra were processed with BrukerTopspin software version 4.0.6.

About 30 mg of sample were dissolved in 3 mL of deuterium oxide (D2O) with 0.002 %TSP and subsequently 0.6 mL were transferred into a 5 mm NMR tube. The HA sample was stirred overnight to ensure a complete solubilization before transfer in an NMR tube (Bruker, Karlsruhe, Germany) for the analysis. <sup>1</sup>H NMR spectra were acquired with pre-saturation of residual HDO, using 64 scans, 12 s relaxation delay, and a number of time-domain points equal to 32k. For the Ch sample, the <sup>1</sup>H-13C HSQC spectra were acquired using 16 scans, 5s relaxation delay, <sup>1</sup> JC-H 150 Hz.

The HA/Ch complex spectra were acquired with a Bruker AVANCE NEO 500MHz spectrometer (Bruker, Karlsruhe, Germany) equipped with a 5 mm TCI cryogenic probe at 303K for <sup>1</sup>H NMR and Diffusion Order NMR Spectroscopy (DOSY). Spectra were processed with Bruker Topspin software version 4.0.6. Ch and HA were dissolved separately in a PBS deuterium volume (mL), which allowed us to reach 7.5 mg/mL and 12.5 mg/mL, respectively. Then, 500 µL of both solutions were taken and mixed, it was vortexed to obtain a homogeneous solution.

Spectra were acquired with pre-saturation of residual HOD using 64 scans, 5 s relaxation delay, and a number of time-domain points equal to 65 k.

DOSY experiments were acquired using the 2D-stimulated echo sequence with bipolar gradient pulse for diffusion. The gradient pulse (δ) and the diffusion time (∆) were set to 5 ms and 300 ms, respectively. The 2D DOSY experiments were run with gradients varied linearly from 5 to 95% in 32 steps, with 16 scans per step. The diffusion coefficients D were extracted using the Bruker Dynamics Center 2.5 (Bruker, Karlsruhe, Germany).
