Profiling Heparan Sulfate-Heavy Metal Ions Interaction Using Electrochemical Techniques

Abstract

Heparan sulfate glycosaminoglycans provides extracellular matrix defense against heavy metals cytotoxicity. Identifying the precise glycan sequences that bind a particular heavy metal ion is a key for understanding those interactions. Here, electrochemical and surface characterization techniques were used to elucidate the relation between the glycans structural motifs, uronic acid stereochemistry, and sulfation regiochemistry to heavy metal ions binding. A divergent strategy was employed to access a small library of structurally well-defined tetrasaccharides analogs with different sulfation patterns and uronic acid compositions. These tetrasaccharides were electrochemically grafted onto glassy carbon electrodes and their response to heavy metal ions was monitored by electrochemical impedance spectroscopy. Key differences in the binding of Hg(II), Cd(II), and Pb(II) were associated with a combination of the uronic acid type and the sulfation pattern.

Keywords: carbohydrates; electrochemistry; heavy metal ions; sulfation pattern; surface chemistry • uronic acid.

Figure 1

a) Scheme of a structurally heterogenous HS polymer; b) amino‐functionalized HS tetrasaccharides with different sulfation and carboxylation.

Scheme 1

Synthesis of G‐tetrasaccharides: a) AgOTf in DCM, −40 °C–RT, 40 min; b) i) NIS, TMSOTf in DCM, −20 °C–RT, 10 min; ii) N₂H₄.H₂O, AcOH in DCM, RT, 3 h; c) i) NIS, TMSOTf in DCM, −20 °C–RT, 10 min; ii) Thiourea in Py/MeOH (1 : 1), 80 °C, 2 h; iii) TEMPO, BAIB in DCM/H₂O (2 : 1), RT, 6 h; iv) MeI, K₂CO₃ in DMF, RT, 12 h; v) Zn dust, THF/AcOH/Ac₂O (3 : 2 : 1), 0 °C, 12 h; d) 70 % HF.py in Pyridine, 0 °C–RT, 24 h; e) DDQ in DCM/H₂O (18 : 1), RT, 2 h; f) SO₃.Me₃N in DMF, 60 °C, 72 h; g) 1 M LiOH in THF/H₂O (1 : 1), RT,18 h; h) H₂, Pd(OH)₂ in H₂O, RT, 36 h.

Scheme 2

Synthesis of I‐tetrasaccharides: a) i) NIS, TMSOTf in DCM, −10 °C–RT, 30 min; ii) Ac₂O, Cu(OTf)₂, RT, 12 h; iii) TMSSPh, ZnI₂, DCM, RT, 2 h; iv) Benzyl (3‐hydroxypropyl)carbamate, NIS, TfOH, RT, DCM, 30 min; b) i) NaOMe, DCM/MeOH (1 : 1), RT, 12 h; ii) TEMPO, DCM/MeOH (1 : 1), RT, 12 h; iii) Zn dust, THF/AcOH/Ac₂O (3 : 2 : 2), RT, 12 h; c) 70 % HF.py in Pyridine, 0 °C–RT, 12 h; d) DDQ in DCM/H₂O (18 : 1), RT, 1 h, 70 %; e) SO₃.Et₃N in DMF, 60 °C, 72 h; f) 1 M LiOH in THF/H₂O (1 : 1), RT, 2 h; g) H₂, Pd(OH)₂ in H₂O, RT, 36 h.

Figure 2

Electrografting of the HS tetrasaccharides onto GC surface, demonstrated by the preparation of I‐6S‐GC; the inset is a representative CV recorded for electrografting of I‐6S.

Figure 3

Impedimetric study of the HS‐GCs response to the heavy metal ions: a) Nyquist plot of I‐6S‐GC response to increasing concentrations of Hg²⁺ ions; b) dose‐response analysis of I‐6S‐GC to increasing concentrations of Pb²⁺, Cd²⁺ and Hg²⁺ ions (N=10); c) impedimetric response of I‐nS‐GC and G‐nS‐GC glycan pairs vs. the sulfation index n in response to 1 μM of the heavy metal ions (only values above the dashed line are considered significant).

Figure 4

Binding preference study of I‐nS and G‐nS glycan pairs towards Cd²⁺ and Hg²⁺ ions represented by the Cd²⁺/Hg²⁺ NR_CT ratio vs. the sulfation index n (measured at the concentration of 1 μM).

Figure 5

Surface characterizations of I‐6S and G‐6S modified GCs prior and after exposure to Cd²⁺ and Hg²⁺ ions: a) CPD analysis (Buffer – after electrografting and exposure to buffer, Cd²⁺/Hg²⁺ – after exposure to 1 μM of each metal ion, ΔCPD values are in respect to the previous step); b) XPS spectra of Hg_4f BE region, and c) Cd_3d BE region (red – I‐6S, blue – G‐6S, doted/solid lines – before/after exposure to 1 μM of the metal ions, grey lines are fit components).