Inova 500

Unless stated otherwise, all reagents and solvents were purchased from commercial suppliers, and all reactions were carried out under anhydrous conditions with argon atmosphere. Yields were calculated by HPLC chromatography or ¹H NMR spectroscopy. UV spectra were obtained using a Perkin-Elmer Lambda2 UV/vis spectrometer. NMR data were collected using either a Varian INOVA 500 (¹H 500 MHz, ¹³C 126 MHz) NMR spectrometer with a 3 mm Nalorac MDBG probe or a Varian INOVA 600 (¹H 600 MHz, ¹³C 150 MHz) NMR spectrometer equipped with a 5 mm ¹H[¹³C,¹⁵N] triple resonance cold probe with a z-axis gradient, utilizing residual solvent signals for referencing. High-resolution mass spectra (HRMS) were obtained using a Bruker (Billerica, MA) APEXII FTICR mass spectrometer equipped with an actively shielded 9.4 T superconducting magnet (Magnex Scientific Ltd., UK), an external Bruker APOLLO ESI source, and a Synrad 50W CO₂ CW laser. Supelco Discover HS (4.6 × 150 mm) and semipreparative (10 × 150 mm) C18 (5 μm) columns were used for analytical and semipreparative HPLC, respectively, as conducted on a Hitachi Elite Lachrom System equipped with a Diode Array L-2455 detector. The enantiomeric excess (ee) was calculated from the area of HPLC peak using a silica-based protein phase enantiomer separation column, RESOLVOSIL BSA-7 column, eluting with 0.1 M phosphate buffer pH 7.5, 2 % 1-propanol.

Norbornene-modified HA was synthesized similar to previous methods.^{33 (link)} Briefly, sodium hyaluronate (Lifecore, 74 kDa) was reacted with Dowex 50W proton-exchange resin, filtered, titrated to pH 7.05, frozen, and lyophilized to yield hyaluronic acid tert-butyl ammonium salt (HA-TBA). HA-TBA was then reacted with 5-norbornene-2-methylamine and benzotriazole-1-yloxytris-(dimethylamino)phosphonium hexafluorophosphate (BOP) in dimethylsulfoxide (DMSO) for 2 h at 25 °C. The reaction was quenched with cold water, dialyzed (molecular weight cutoff: 6–8 kDa) for 5 days, filtered, dialyzed for 5 more days, frozen, and lyophilized. The degree of modification was 22% as determined by ¹H NMR (500 MHz Varian Inova 500, Figure S1).

Unless stated otherwise, all reagents and solvents were purchased from commercial suppliers. Yields were calculated by HPLC chromatography or ¹H NMR spectroscopy. Circular dichroism spectra were obtained on a Jasco J720A spectropolarograph. NMR data were collected using either a Varian INOVA 500 (¹H 500 MHz, ¹³C 126 MHz) NMR spectrometer with a 3 mm Nalorac MDBG probe or a Varian INOVA 600 (¹H 600 MHz, ¹³C 150 MHz) NMR spectrometer equipped with a 5 mm ¹H[¹³C,¹⁵N] triple resonance cold probe with a z-axis gradient, utilizing residual solvent signals for referencing. High-resolution mass spectra (HRMS) were obtained using a Bruker (Billerica, MA) APEXII FTICR mass spectrometer equipped with an actively shielded 9.4 T superconducting magnet (Magnex Scientific Ltd., UK), an external Bruker APOLLO ESI source, and a Synrad 50W CO₂ CW laser. Supelco Discover HS (4.6 × 150 mm) and semipreparative (10 × 150 mm) C₁₈ (5 μm) columns were used for analytical and semipreparative HPLC, respectively, as conducted on a Hitachi Elite Lachrom System equipped with a Diode Array L-2455 detector.

The NMR data were all collected at 20 °C on a double-labeled (¹³C, ¹⁵N) sample (~1 mM) of P₅S₃ using four-channel Varian Inova-500, -600 and -750 spectrometers equipped with triple resonance probes and pulse field gradients. Two-dimensional ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra were obtained under a variety of buffer conditions similar to those used for the CD studies, however, 2% acetic acid (2% CD₃CO₂D, 7% D₂O, 91% H₂O) was chosen for the collection of backbone assignment NMR data because the quality of the data was best under this condition. This data consisted of standard two-dimensional ¹H-¹⁵N and ¹H-¹³C HSQC experiments and three-dimensional HNCACB, HNCA, CC-TOCSY-NNH, ¹⁵N-edited NOESY (90 ms mixing time), and HNN experiments using the Varian Biopack suite of pulse programs. The average backbone amide ¹⁵N T₁/T_1ρ ratios, measured with a modified ¹H-¹⁵N HSQC experiment to record one-dimensional spectra,^{60 (link),61 (link)} was used to estimate an overall rotational correlation time (τ_c) for P₅S₃ at 20 °C. Felix2007 (MSI, San Diego, CA) and Sparky (v3.115) were used to process and analyze, respectively, all the NMR data. Indirect methods (DSS = 0 ppm) were used to reference the ¹H, ¹³C, and ¹⁵N chemical shifts.^{62 (link)}

All chemicals were purchased from commercial sources as noted below and used as received. NMR spectra were recorded on a Varian Inova 500 (500 MHz for ¹H and 125 MHz for ¹³C) instrument in CD₃OD and were recorded at ambient temperatures. Chemical shifts are reported in units of parts per million (ppm). ¹H NMR were calibrated using the residual protio-solvent as a standard. ¹³C NMR spectra are calibrated using the deutero-solvent as a standard. High-resolution mass spectra (HRMS) was obtained on a Bruker micrOTOFQ-II mass spectrometer.
o-AMSA hydrochloride was prepared from 9-chloroacridine (Aldrich) and N-(4-amino-2-methoxyphenyl)methanesulfonamide (ChemBridge) according to a literature procedure^{41 (link)}. ¹H NMR (500 MHz, CD₃OD): δ 8.23 (ddd, J = 8.8, 1.3, 0.6 Hz, 2H), 8.03 – 7.98 (m, 2H), 7.95 (ddd, J = 8.6, 1.3, 0.6 Hz, 2H), 7.55 (d, J = 8.4 Hz, 1H), 7.48 (ddd, J = 8.8, 6.7, 1.3 Hz, 2H), 7.17 (d, J = 2.3 Hz, 1H), 7.02 (dd, J = 8.4, 2.3 Hz, 1H), 3.84 (s, 3H), 3.02 (s, 3H); ¹³C NMR (126 MHz, CD₃OD): δ 157.3, 154.1, 141.8, 139.7, 136.9, 127.6, 126.8, 125.9, 125.4, 120.2, 118.1, 115.2, 109.5, 56.8, 39.9; HRMS (m/z): [M]⁺ calcd. for [C₂₁H₂₀N₃O₃S]⁺, 394.1220; found, 394.1224. see Supplementary Fig. 12 for NMR spectra.