Topsin 2

LMWH samples were analyzed by ¹H, ¹³C nuclear magnetic resonance (NMR), and two-dimensional NMR spectroscopy. Heteronuclear single-quantum coherence (HSQC), proton–proton correlation spectroscopy (HHCOSY) and total correlation spectroscopy (TOCSY) were used to characterize theirstructures.^{2 ,17 (link)} All NMR experiments were performed on Bruker Advance II 600 MHz spectrometer (Bruker Bio Spin, Billerica, MA) with Topsin 2.1.6 software (Bruker). Samples were each dissolved in 0.5 mL ²H₂O (99.996 %, Sigma Chemical Company) and freeze-dried repeatedly to remove the exchangeable protons. The samples were redissolved in 0.4 mL ²H₂O and transferred to NMR microtubes (outside diameter, 5 mm, Norell (Norell, Landisville, NJ)). The conditions for one-dimensional ¹H-NMR spectra were as follows: wobble sweep width of 12.3 kHz, acquisition time of 2.66 s, and relaxation delay of 8.00 s. Temperature was 298 K. The conditions for two-dimensional HSQC spectra were as follows: 32 scans, sweep width of 6.15 kHz, acquisition time of 0.33 s, and relaxation delay of 0.90 s. The conditions for two-dimensional HHCOSY spectra were as follows: 16 scans, sweep width of 7.46 kHz, acquisition time of 0.28 s, and relaxation delay of 1.50 s.^{16 (link)}

Heparosan and heparin products were analyzed by ¹H nuclear magnetic resonance NMR). All NMR experiments were performed on a Bruker Advance II 600 MHz spectrometer (Bruker BioSpin, Billerica, MA) with Topsin 2.1.6 software (Bruker) as previously reported.^{40 (link),41 (link)} Briefly, samples were each dissolved in 0.5 mL D₂O (99.96%, Sigma) and freeze-dried twice to remove the exchangeable protons. The samples were re-dissolved in 0.4 mL D₂O and transferred to NMR micro tubes (OD 5 mm, Norell, tubes). ¹H NMR experiments were performed with 64 scans and an acquisition time of 1.5 sec at 298 K.