Functional chitooligosaccharide synthesis
COS was prepared from chitosan through nitrous acid
depolymerization (shown in Figure 2) based on the protocols
by Moussa [24, 26]; low MW chitosan was dissolved in 0.15 M HCl
(2 g chitosan per 100 mL solution), then vacuum degassed to
remove oxygen from the solution. 500 mM NaNO2 aqueous
solution was prepared (with degassed MilliQ water,
immediately before addition to chitosan solution), which was
then added for a GlcN:NaNO2 molar ratio of 10:1 (to achieve
COS with approximately 20 average GlcN units), before being
stirred moderately overnight so as not to introduce excess oxygen
to the mixture. The mixture was filtered to remove undissolved
chitosan and other impurities, before precipitating COS by
addition of ammonium hydroxide until reaching solution pH 9.
To further increase yield, an equal volume of methanol was added
to the mixture, which was then centrifuged for 10 min at 10,000 g
and 4°C. The COS was washed (x3) by repeated centrifugation in
50/50 water/methanol, before a final wash step with water. The
COS was then resuspended in 10 mL water and lyophilized.
Hydrazide functionalized COS (COS-hydrazide) was prepared
by dissolving COS in water, with additions of acetic acid.
Concentrated MES was then added to a concentration of
0.15 M, before adjusting the solution to pH 4.5 with NaOH or
HCl. 10 M equivalents (to COS) of adipic dihydrazide was added to
solution and stirred at RT for 24 h, then 10 M equivalents (to COS)
of sodium cyanoborohydride was added to solution and stirred at
RT for 24 h. As depicted in Figure 3, the COS aldehyde functional
group reacts with the hydrazide under acidic conditions to form a
hydrazone conjugate, which is further reduced to a secondary amine
bond by sodium cyanoborohydride. The COS-hydrazide solution
was filtered, precipitated, washed and lyophilized following the same
protocol as COS. COS and COS-hydrazide were analysed with 1 H
NMR spectroscopy to confirm average size and presence of
aldehyde or hydrazide groups, respectively.
1
H NMR
spectroscopy was performed on Bruker 500 MHz spectrometer
at 300 K. Samples were diluted in deuterated solvent (D2O) to
10 mg/mL; 5 μL HCl (12 M) was added to 1000 μL COS samples to
facilitate dissolution in D2O. NMR spectra was analysed using
Bruker TopSpin NMR data analysis software.