Kinetics of Calcite Nucleation onto Sulfated Chitosan Derivatives and Implications for Water-Polysaccharide Interactions during Crystallization of Sparingly Soluble Salts

Abstract

Anionic macromolecules are found at sites of CaCO₃ biomineralization in diverse organisms, but their roles in crystallization are not well-understood. We prepared a series of sulfated chitosan derivatives with varied positions and degrees of sulfation, DS(SO₃ ^-), and measured calcite nucleation rate onto these materials. Fitting the classical nucleation theory model to the kinetic data reveals the interfacial free energy of the calcite-polysaccharide-solution system, γ_net, is lowest for nonsulfated controls and increases with DS(SO₃ ^-). The kinetic prefactor also increases with DS(SO₃ ^-). Simulations of Ca²⁺-H₂O-chitosan systems show greater water structuring around sulfate groups compared to uncharged substituents, independent of sulfate location. Ca²⁺-SO₃ ^- interactions are solvent-separated by distances that are inversely correlated with DS(SO₃ ^-) of the polysaccharide. The simulations also predict SO₃ ^- and NH₃ ⁺ groups affect the solvation waters and HCO₃ ^- ions associated with Ca²⁺. Integrating the experimental and computational evidence suggests sulfate groups influence nucleation by increasing the difficulty of displacing near-surface water, thereby increasing γ_net. By correlating γ_net and net charge per monosaccharide for diverse polysaccharides, we suggest the solvent-separated interactions of functional groups with Ca²⁺ influence thermodynamic and kinetic components to crystallization by similar solvent-dominated processes. The findings reiterate the importance of establishing water structure and properties at macromolecule-solution interfaces.

Figure 1

Natural chitin is deacetylated by alkaline hydrolysis to yield (A_i) chitosan (control). This material was derivatized by two methods to prepare (A_ii) O-sulfated chitosan (where most sulfate groups (R = SO₃^–) are on the C₆O-position (solid circle) but can also be present at the C₃O-position (dashed)) and (A_iii) N-sulfated chitosan. ¹H NMR spectra of (B_i) chitosan, (B_ii) O-sulfated chitosan, and (B_iii) N-sulfated chitosan show the characteristic proton peaks used to quantify DS(Ac).

Figure 2

Representative kinetic data for chitosan material OSC DS(SO₃^–) = 0.42. (A) Optical image collected at 1 h of reaction time shows calcite crystallites (with postnucleation growth); (B) slope of the number of nuclei formed per area versus time is determined to obtain the nucleation rate, J₀, from separate experiments conducted at each saturation state, σ (eq 8). The dependence of rate on 1/σ² yields interfacial free energy, γ_net (e.g., eq 14 and Figure 3).

Figure 3

Experimental data show slope, B, is composition specific. Chitosan A and B are cross-linked (XL) and not-XL, respectively. The B values are determined for each experiment (±1 standard error) using eq 14: (A) B_{chitosan A(Control, XL)} = 148 ± 74; (B) B_{chitosan B(Control, not XL)} = 186 ± 11; (C) B_OSC 0.23 = 171 ± 30; (D) B_OSC 0.42 = 182 ± 10; (E) B_OSC 0.77 = 273 ± 86; (F) B_NSC 0.13 = 162 ± 81; (G) B_NSC 0.28 = 217 ± 172; (H) B_NSC 0.47 = 230 ± 47. All data were collected at pH 10, 22 °C.

Figure 4

Analysis of kinetic data shows that (A) B (recall B ∝ γ_net, eq 12) increases with sulfate density, DS(SO₃^–), but is otherwise approximately independent of composition. (B) Kinetic prefactor, A, also increases with DS(SO₃^–). The standard error of B and ln(A) are determined from the fit of eq 14 to the data in Figure 3.

Figure 5

(A) RDF profiles for O-sulfated chitosans. Sulfate groups create a region of high-water structuring ≈3.9 Å of S–H₂O separation (black) relative to uncharged substituents (gray). Charged amines also structure water (green). The model predicts Ca²⁺ ions are associated with sulfate in the lower-water density region ≈5 Å from the sulfate groups (compare blue and black lines), indicating solvent-separated Ca²⁺–sulfate interactions. A second region of Ca²⁺ is present ≈9 Å from sulfate groups. (B) RDF profiles for N-sulfated chitosans also show sulfate groups promote regions of high-water density. Ca²⁺ shows a greater separation distance of ≈7.5 Å from sulfate groups and a broader distribution (red). (C) Most probable distance between sulfated group and Ca²⁺ is inversely correlated with DS(SO₃^–), and independent of sulfate position. (D) Smaller Ca²⁺–S separation distance associated with the high DS(SO₃^–) is correlated with a high energy barrier to nucleation by a general relationship that is independent of sulfate position. (E) Number of waters about Ca²⁺ increase as DS(SO₃^–) increases (Ca²⁺–S separation declines), consistent with predictions of the solvation sphere about Ca²⁺–SO₄^2– ion pairs. (F) Number of bicarbonate ions associated with Ca²⁺ decreases as DS(SO₃^–) increases but appears to be position-dependent. (G) Number of waters about Ca²⁺ decreases with increasing DS(NH₃⁺) potentially due to a correlation with HCO₃^–. (H) Number of bicarbonate ions associated with Ca²⁺ increases as DS(NH₃⁺) increases but this factor alone cannot explain all values.

Figure 6

Energy barrier to calcite nucleation correlates with net charge per monosaccharide by a general relationship that includes diverse polysaccharide compositions. The dependence includes values reported by Guiffre et al., for heparin, hyaluronic acid (HA), and two alginates with high (HG-Alg) or low (LG-Alg) guluronic acid content. Tests of carboxymethyl chitosan (CMCS, Figure 7) also agree with the trend. Values of γ_net (mJ m^–2) shown here: γ_{net Chitosan A(Control, XL)} = 57; γ_{net Chitosan B(Control, not XL)} = 59; γ_{net NSC 0.13} = 54; γ_{net NSC 0.28} = 62; γ_{net NSC 0.47} = 63; γ_{net OSC 0.23} = 57; γ_{net OSC 0.42} = 58; γ_{net OSC 0.77} = 66; and γ_net CMCS = 72. And: γ_{net Chitosan(Giuffre)} = 51; γ_{net HA(Giuffre)} = 65; γ_{net HG-Alg(Giuffre)} = 69; γ_{net LG-Alg(Giuffre)} = 76, γ_{net Heparin(Giuffre)} = 75. Values were obtained using eq 12 and F = 16/3π, ω = 6.13 × 10^–23 cm³, and T = 22 and 25 °C for data reported in this study and Giuffre et al., respectively.

Figure 7

(A) Carboxymethyl chitosan (CMCS, prepared and characterized by Zhou et al.). (B) Calcite nucleation rate measurements yield B_CMCS = 346 ± 99. (C) MD simulations show carboxyl groups also create regions of high-water structuring with a magnitude that is greater than sulfate but dependent on carboxyl position.