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The complexation of 6 bile salts with various methylated β-cyclodextrins was studied to elucidate how the degree and pattern of substitution affects the binding. The structures of the CDs were determined by mass spectrometry and NMR techniques, and the structures of the inclusion complexes were characterized from the complexation-induced shifts of (13)C nuclei as well as by 2D ROESY NMR. Thermodynamic data were generated using isothermal titration calorimetry. The structure-properties analysis showed that methylation at O3 hinders complexation by partially blocking the cavity entrance, while methyl groups at O2 promote complexation by extending the hydrophobic cavity. Like in the case of 2-hydroxypropylated cyclodextrins, the methyl substituents cause an increased release of ordered water from the hydration shell of the bile salts, resulting in a strong increase in both the enthalpy and the entropy of complexation with increased number of methyl substituents. Due to enthalpy-entropy compensation the effect on the stability constant is relatively limited. However, when all hydroxyl groups are methylated, the rigid structure of the free cyclodextrin is lost and the complexes are severely destabilized due to very unfavorable entropies.

Three series of novel water-soluble beta-cyclodextrin-dextran polymers have been prepared by "click" chemistry. The polymers were synthesized from alkyne-modified dextrans (AMDs) onto which mono-6-O-deoxy-monoazido-betaCD (N3betaCD) was grafted by a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The polymers have been characterized by NMR spectroscopy and size exclusion chromatography (SEC). The binding properties have been characterized by isothermal titration calorimetry (ITC) and show excellent accessibility of the betaCDs.

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Many therapeutic proteins require storage at room temperature for extended periods of time. This can lead to aggregation and loss of function. Cyclodextrins (CDs) have been shown to function as aggregation suppressors for a wide range of proteins. Their potency is often ascribed to their affinity for aromatic amino acids, whose surface exposure would otherwise lead to protein association. However, no detailed structural studies are available. Here we investigate the interactions between human growth hormone (hGH) and different CDs at low pH. Although hGH aggregates readily at pH 2.5 in 1 M NaCl to form amorphous aggregates, the presence of 25 to 50 mM of various ␤-CD derivatives is sufficient to completely avoid this. ␣-and ␥-CD are considerably less effective. Stopped-flow data on the aggregation reaction in the presence of ␤-CD are analyzed according to a minimalist association model to yield an apparent hGH-␤-CD dissociation constant of ∼6 mM. This value is very similar to that obtained by simple fluorescence-based titration of hGH with ␤-CD. Nuclear magnetic resonance studies indicate that ␤-CD leads to a more unfolded conformation of hGH at low pH and predominantly binds to the aromatic side-chains. This indicates that aromatic amino acids are important components of regions of residual structure that may form nuclei for aggregation.

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The effect of the degree of substitution (DS) on the ability of hydroxypropylated β-cyclodextrin (HPβCD) to form inclusion complexes with six different bile salts, found within the intestinal tracts of rats, dogs, and humans, was studied by isothermal titration calorimetry. The composition and molecular structure of the cyclodextrin samples were characterized by MALDI-TOF mass spectrometry together with 1D and 2D-NMR, and some of the complexes were studied by 2D ROESY NMR. The stability and structure of the complexes were mainly determined by the position of hydroxyl groups on the bile salts and depended relatively little on the number of hydroxypropyl side chains on the CDs. The enthalpy and entropy of complexation exhibited a strong linear increase as the DS increased from 0 to 1, and a pronounced enthalpy-entropy compensation was observed. These observations are interpreted as an increased release of ordered water from the hydration shells of the bile salts, caused by the hydroxypropyl substituents on the rim of the CD. It is estimated that each CD hydroxypropyl substituent dehydrates a hydrophobic surface area of approximately 10 Å(2).