Modulating Thiol pK a Promotes Disulfide Formation at Physiological pH: An Elegant Strategy To Design Disulfide Cross-Linked Hyaluronic Acid Hydrogels

The disulfide bond plays a crucial role in protein biology and has been exploited by scientists to develop antibody–drug conjugates, sensors, and for the immobilization other biomolecules to materials surfaces. In spite of its versatile use, the disulfide chemistry suffers from some inevitable limitations such as the need for basic conditions (pH > 8.5), strong oxidants, and long reaction times. We demonstrate here that thiol-substrates containing electron-withdrawing groups at the β-position influence the deprotonation of the thiol group, which is the key reaction intermediate in the formation of disulfide bonds. Evaluation of reaction kinetics using small molecule substrate such as l-cysteine indicated disulfide formation at a 2.8-fold higher (k 1 = 5.04 × 10–4 min–1) reaction rate as compared to the conventional thiol substrate, namely 3-mercaptopropionic acid (k 1 = 1.80 × 10–4 min–1) at physiological pH (pH 7.4). Interestingly, the same effect could not be observed when N-acetyl-l-cysteine substrate (k 1 = 0.51 × 10–4 min–1) was used. We further grafted such thiol-containing molecules (cysteine, N-acetyl-cysteine, and 3-mercaptopropionic acid) to a biopolymer namely hyaluronic acid (HA) and determined the pK a value of different thiol groups by spectrophotometric analysis. The electron-withdrawing group at the β-position reduced the pK a of the thiol group to 7.0 for HA-cysteine (HA-Cys); 7.4 for N-acetyl cysteine (HA-ActCys); and 8.1 for HA-thiol (HA-SH) derivatives, respectively. These experiments further confirmed that the concentration of thiolate (R-S–) ions could be increased with the presence of electron-withdrawing groups, which could facilitate disulfide cross-linked hydrogel formation at physiological pH. Indeed, HA grafted with cysteine or N-acetyl groups formed hydrogels within 3.5 min or 10 h, respectively, at pH 7.4. After completion of cross-linking reaction, both gels demonstrated a storage modulus G′ ≈ 3300–3500 Pa, which indicated comparable levels of cross-linking. The HA-SH gel, on the other hand, did not form any gel at pH 7.4 even after 24 h. Finally, we demonstrated that the newly prepared hydrogels exhibited excellent hydrolytic stability but can be degraded by cell-directed processes (enzymatic and reductive degradation). We believe our study provides a valuable insight on the factors governing the disulfide formation and our results are useful to develop strategies that would facilitate generation of stable thiol functionaliz