Influence of sulfate and carboxylate groups on the conformation of chondroitin sulfate related disaccharides.

Abstract 1 H NMR and 13 C NMR spectra parameters of eight sulfated uronic acid containing disaccharides 1–8 were used to determine the conformation preferences that depend on the pattern of sulfation. Three sulfated derivatives of benzyl β- d -Gal-(1 → 4)-β- d -GlcA ( 1 ), its 6′-sulfate 2 , 4′-sulfate 3 , and 4′,6′-disulfate 4 were used as models for the β-(1 → 4) glycosidic linkage of chondroitin sulfates and three sulfated derivatives of benzyl β- d -GlcA-(1 → 3)-β- d -Gal ( 5 ), its 6-sulfate 6 , 4-sulfate 7 , and 4,6-disulfate 8 were used as models for the β-(1 → 3) glycosidic linkage of chondroitin sulfates. To determine the dependence of conformational preferences on the charged groups, the sulfated disaccharides 2 , 3 , and 4 were compared to their unsulfated parent compound 1 , and 6 , and 7 , and 8 were compared to their parent compound 5 . The 3 J H-5,H-6 coupling constants were determined by high-order analysis of the spin systems, and from these the preferred populations of the hydroxymethyl groups were calculated. Selective 1D NOEs and ROEs were measured from H-1′ across the glycosidic linkage to obtain the average distance of the protons adjacent to the glycosidic linkage. Derivatives of β- d -Gal-(1 → 4)-β-GlcA carrying a sulfate group in the 6t-position ( 2 ) and in the 4′- and 6′-position ( 4 ) show a slight repulsive effect between the 6′-slfate groups and the carboxylate group as expressed in small changes of the preferred population of the glycosidic linkage and the sulfonyloxymethyl group. The 4-sulfate groups in 3 and 4 do not show a significant influence on the glycosidic linkage. However, the two sulfate groups in 4 exhibit a repulsive effect leading to a very high population of the gt conformation of the sulfonyloxymethyl group. In contrast hereto, the sulfate group at C-4 of β- d -GlcA- (1 → 3)-β- d -Gal disaccharides 7 and 8 and the carboxylate group exert an attractive interaction that leads to a change of the conformation of the glycosidic linkage in 7 and 8 by about 30°. The 6-sulfate groups of the disaccharides 6 and 8 show a slight repulsive interaction with the carboxylate and/or 4-sulfate group. Changes in 13 C NMR chemical shifts support the interpretation obtained from the NOE and ROE analysis. Relative NOE ratios were used to estimate the correlation time to τ c = 3.5-4.0x10 −10 s for 1–8 , and these were found to be in agreement with calculated T 1 values. The charge-charge interaction energies observed are fairly small in 1–8 . The largest effect can be observed in the rotamer population of the C-6 groups of the disulfates 4 and 8 , which leads to an effective interaction energy of the two sulfate groups of ≈1.0 kcal/mol. This is equivalent to an effective dielectric constant of ∈ ≈ 10, assuming that full charges are expressed on the sulfate groups. Ensemble average values for observable ∈ of 1–8 were obatined from Metropolis Monte Carlo simulations (MMC) that were run with dielectric constant ∈ ≈ 2–80 using the GEGOP program. The results of the calculations agree well with the experimental data at dielectric constants of ∈ ≈ 10.