Nonpolar Cavity Research Articles

Beta-cyclodextrin induced room temperature phosphorescence from 1-bromonaphthalene in the presence of naphthalene and 1-butanol

Intense room temperature phosphorescence (RTP) from host-guest inclusion complex of beta-cyclodextrin (β-CD) with 1-bromonaphthalene (1-BrN), stabilized by naphthalene (N) and 1-butanol (B), has been investigated. The spectral characteristics of luminescence emission and optimal conditions involved are reported. It has been confirmed that N and B are incorporated into the nonpolar cavity of β-CD as the second and third guests. The extra space inside the cavity is more filled by N and B, and δ-CD more effectively shields 1-BrN from quencher in aqueous solution. B completely hinders the formation of N excimer. Greater rigidity of the complex was achieved. Consequently, much more intense fluorescence and phosphorescence appear in comparison with the ternary β-CD:1-BrN:B complex. The limit of detection is 7.38 × 10 −8mol 1 −1 for 1-BrN and 1.36 × 10 −6mol 1 −1 for N.

Energetic origins of specificity of ligand binding in an interior nonpolar cavity of T4 lysozyme.

To determine the constraints on interactions within the core of a folded protein, we have analyzed the binding of 91 different compounds to an internal cavity created in the interior of phage T4 lysozyme by site-directed mutagenesis [Eriksson et al. (1992a) Nature 355, 371-373]. The cavity is able to accommodate a variety of small, mainly nonpolar, ligands. Molecules which do not appear to bind include those that are very polar, those that are too large, and those that have appropriate volume and polarity but inappropriate shape. Calorimetric analysis of 16 of these ligands reveals that their free energies of binding are poorly correlated with their solvent-transfer free energies. In addition, their enthalpies of binding are much larger than expected on the basis of transfer of the ligands from an aqueous to a nonpolar liquid phase. The binding energetics were analyzed by dividing the reaction into three processes: desolvation, immobilization, and packing. This analysis indicates that all three processes contribute to binding specificity. For a subset of these ligands that are structurally related, however, packing interactions in the protein interior are well modeled by the interactions of the ligands with octanol.

Specificity of ligand binding in a buried nonpolar cavity of T4 lysozyme: Linkage of dynamics and structural plasticity

To better understand the role of shape complementarity in ligand binding and protein core interactions, the structures have been determined of a set of ligands bound within a cavity-containing mutant of T4 lysozyme. The interior cavity is seen to consist of two parts that respond very differently to the binding of ligands. First, there is a relatively rigid region that does not relax significantly upon binding any ligand. Second, there is a more flexible region that moves to various extents in response to binding the different ligands. The part of the binding site that remains rigid is characterized by low temperature factors and strong protection from hydrogen exchange. This part of the site appears to be primarily responsible for discriminating between ligands of different shape (i.e., for determining specificity). The more flexible region, characterized by relatively high temperature factors and weak protection from hydrogen exchange, allows some promiscuity in binding by undergoing variable amounts of deformation at essentially the same energetic cost. This linkage between the dynamic information represented by crystallographic temperature factors and hydrogen-exchange behavior on the one hand, and structural plasticity in response to ligand binding on the other hand, suggests a way to improve our understanding of steric interactions in protein cores and protein-ligand binding sites. Ligand design and packing algorithms might take advantage of this information, requiring complementary interactions where the protein is rigid and allowing some overlap in regions where the protein is flexible.

On the Probability of Finding a Water Molecule in a Nonpolar Cavity

Measurements of solubility and vapor pressure indicate that the cost of making a cavity in a nonpolar solvent that is large enough to accommodate a single water molecule is just balanced by the attraction between the water molecule and the walls of the cavity. As a result of this unexpected coincidence, the equilibrium constant for entry of an isolated water molecule from the vapor phase into cyclohexane is almost exactly unity at room temperature. Molecules smaller than water prefer the vapor phase, whereas molecules larger than water prefer cyclohexane. In equilibrium with liquid water, the tendency of single water molecules to enter small nonpolar cavities, such as may be present in mutant proteins, is expected to be vanishingly small.

Binding of 2-Acetylnaphthalene to Cyclodextrins Studied by Fluorescence Quenching

The molecule 2-acetylnaphthalene (2-AN) exhibits significant room-temperature fluorescence only in strong hydrogen-bonding solvents, such as water and fluorinated alcohols. As a consequence, the fluorescence of 2-AN is strongly quenched when it binds in the relatively nonpolar cavity of cyclodextrins (CDs). Fluorescence lifetime data confirm that the quenching is static rather than dynamic, and thus the slope of the Stern-Volmer quenching plot (F°/F vs . [CD]) is equal to K, the binding constant. We have used these plots to measure K for 1:1 complexes formed between 2-AN and several CDs, including unsubstituted α;-, β-, and γ-CD and a dimethyl substituted β-CD. In addition, we have determined Δ H° and Δ S° of binding from the temperature dependence of these plots. The room-temperature binding constants vary over a 50-fold range for the 2-AN:CD complexes studied herein, and the variability stems primarily from the Δ S° term. We report here the results of this work and also show evidence for observations other than 1:1 complexes, including emission from the excimer of 2-AN in the presence of γ-CD.

High-performance liquid chromatographic enantioseparation of glycyl di- and tripeptides on native cyclodextrin bonded phases: Mechanistic considerations

Di- and tripeptides containing at least one glycine moiety were separated into enantiomers on χ-, β- and γ-cyclodextrin bonded phases after their precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). It is shown that the choice of a suitable cyclodextrin bonded phase used with a nonaqueous polar mobile phase offers a wide range of possibilities to optimize enantioselectivity. Use of the FMOC derivative greatly enhances sensitivity, stability and enantioselectivity.It is postulated that under reversed-phase conditions the inclusion complex formation between the hydrophobic aromatic part of the FMOC-functionalized peptides and non-polar cyclodextrin cavity interior is the major factor contributing to retention. However, under these conditions there seems to be insufficient interaction between the hydrophilic peptide chain and cyclodextrin hydroxyls. Hence no chiral recognition and enantiomeric separation is observed. In systems operating with polar organic mobile phases the inclusion complex is suppressed as the cyclodextrin cavity is largely occupied by the mobile phase. The enantioselectivity observed is caused by hydrogen bonding between peptide chain and the hydroxyl groups at the mouth of the cyclodextrin. The stereospecific interactions depend very strongly on the cyclodextrin size, the length of the peptide chain and the mobile phase composition.

12,52,92,132-Tetranitro-1,5,9,13(1,3)-tetrabenzena-3,7,11,15(1,4)-tetrapiperazinacyclo-hexadecaphane, a new host compound

The new 36-membered macrocyclic host molecule 12,52,92,132-tetranitro-1,5,9,13(1,3)-tetrabenzena-3,7,11,15(1,4)-tetrapiperazinacyclohexadecaphane, has been synthesised by a cyclisation reaction of 2,6-bis(bromomethyl)nitrobenzene and piperazine with Na2CO3 as a base under high dilution conditions; the macrocycle has an ellipsoidal non-polar cavity with a diameter of ca. 8 A and has been characterized as a 1 : 1 inclusion complex with acetonitrile by X-ray diffraction and 1H NMR.

Steroid-binding site of human and rabbit sex steroid binding protein of plasma: fluorescence characterization with equilenin.

The interaction of the estrogen d-3-hydroxy-1,3,5(10),6,8-estrapentaen-17-one (equilenin) with the human and rabbit sex steroid binding proteins (hSBP and rSBP, respectively) has been investigated by using fluorescence and absorption spectroscopy. Equilenin competes for the binding of 5 alpha-dihydrotestosterone. The calculated binding constant of equilenin for rSBP is 1.9 X 10(7) M-1 at 4 degrees C, which can be compared with the binding constant of 5.7 X 10(7) M-1 reported for hSBP [Ross, J.B.A., Torres, R., & Petra, P.H. (1982) FEBS Lett. 149, 240]. The results of fluorescence quenching experiments with the collisional quenchers KI and acrylamide indicate that the bound steroid has limited accessibility to the bulk solvent and that there are no anionic surface groups near the steroid-binding site. The fluorescence excitation spectra of SBP-equilenin complexes are similar to the absorption spectra of equilenin in low-dielectric solvents. The fluorescence emission of the SBP-equilenin complexes, however, exhibits wavelength shifts (red shifts) opposite to those of the steroid in low-dielectric solvents or complexed with beta-cyclodextrin (blue shifts) but similar to the red shift produced by addition of the proton acceptor triethylamine to equilenin in cyclohexane. These data indicate that the steroid-binding site of hSBP and rSBP is a nonpolar cavity containing a proton acceptor that participates in a specific interaction, possibly a hydrogen bond, with the 3'-hydroxyl group of the bound steroid.

Intramolecular Interactions in the Ground and Excited States of 1,3-Bichromophoric Propanes in the Aqueous Cyclodextrin Solutions

Abstract The enhanced fluorescence of several 1,3-bichromophoric propanes in aqueous cyclodextrin solutions has been investigated by the steady-state fluorescence spectra and fluorescence lifetimes. 1-(9,10-Dicyano-2-anthryl)-3-(1- and 2-naphthyl)propanes exhibit the intramolecular charge transfer complex fluorescence generated in the ground state in the presence of γ-cyclodextrin. Two bichromophoric propanes, 1,3-di-2-naphthylpropane and 1-(1-naphthyl)-3-(2-naphthyl)propane, in the aqueous β- and γ-cyclodextrin solutions show not only the intramolecular excimer but also the ground state complex fluorescences, though the ground state complex formation of dinaphthylpropane has never been observed in the homogeneous solution even at low temperature. The intramolecular interactions in the ground and excited states seem to be attributable to their intramolecularly geometrical restriction in the extraordinary nonpolar cavity of the cyclodextrins.

Structure of crystalline α-chymotrypsin: II. A preliminary report including a hypothesis for the activation mechanism

An electron density map of tosyl-α-chymotrypsin at 2 A resolution is presented, which shows the conformation of the polypeptide chain. An electron density map of the differences between the tosylated and the native enzyme has also been calculated. These maps lead to the following conclusions. Histidine 57 and serine 195 are known to be part of the active site. In the native enzyme, these are in an environment open to the solvent and in a conformation consistent with the existence of a hydrogen bond between them. In the tosylated enzyme, small movements of histidine 57, serine 195 and methionine 192 bring histidine 57 into a position where it can interact with the sulphonyl group. No other conformational changes are observed. The enzyme contains an ion pair between the α-amino group of isoleucine 16 and the β-carboxyl group of aspartate 194, located in an otherwise non-polar cavity. In conjunction with kinetic and spectroscopic studies (especially Oppenheimer, Labouesse & Hess, 1966) and X-ray analysis of chymotrypsinogen and δ-chymotrypsin at low resolution (Kraut, Sieker, High & Freer, 1962; Kraut, Wright, Kellerman & Freer, 1967), our results lead to a hypothesis for the stereochemistry of the activation process. It is proposed that (i) activation of the zymogen involves no gross reorganization of the main chain nor a significant helix-coil transition; (ii) activation involves a structural change of the enzyme, caused by the formation to an ion-pair between isoleucine 16 and aspartate 194. This structural change would be reversed when the positively charged α-amino group of isoleucine 16 is deprotonated at high pH. The reversal seems to be sterically blocked when the enzyme is inhibited by a bulky group on serine 195. In the light of our model, the location of the disulphide bridges in the chemical sequence of trypsin suggests that trypsin and chymotrypsin have nearly identical tertiary structures, and that their disulphide bridges serve to stabilize rather than to determine the structure. The interactions between the molecules related by the non-crystallographic dyad axes in the crystal are described. One of these involves the active site, but does not inhibit enzymic activity. Of the heavy atoms used for phase determination by the method of isomorphous replacement, platinum(II)- and mercury-containing substituents are bound to sites containing cystine and methionine residues.