Chiral Recognition of Dansyl Derivatives with an Amino Acid-Based Molecular Micelle: A Molecular Dynamics Investigation.

In this study, the chiral separation mechanisms of Dansyl amino acids, including Dansyl-Leucine (Dans-Leu), Dansyl-Norleucine (Dans-Nor), Dansyl-Tryptophan (Dans-Trp) and Dansyl-Phenylalanine (Dans-Phe) binding to poly-sodium N-undecanoyl-(L)-Leucylvalinate, poly(SULV), were investigated using molecular dynamics simulations. Micellar electrokinetic chromatography (MEKC) has previously shown that when separating the enantiomers of these aforementioned Dansyl amino acids, the L- enantiomers bind stronger to poly(SULV) than the D- enantiomers. This study aims to investigate the molecular interactions that govern chiral recognition in these systems using computational methods. This study reveals that the computationally-calculated binding free energy values for Dansyl enantiomers binding to poly(SULV) are in agreement with the enantiomeric order produced in experimental MEKC studies. The L- enantiomers of Dans-Leu, Dans-Nor, Dans-Trp, and Dans-Phe binding to their preferred binding pockets in poly(SULV) yielded binding free energy values of -21.8938, -22.1763, -21.3329 and -13.3349 kJ·mol-1, respectively. The D- enantiomers of Dans-Leu, Dans-Nor, Dans-Trp, and Dans-Phe binding to their preferred binding pockets in poly(SULV) yielded binding free energy values of -14.5811, -15.9457, -13.6408, and -12.0959 kJ·mol-1, respectively. Furthermore, hydrogen bonding analyses were used to investigate and elucidate the molecular interactions that govern chiral recognition in these molecular systems.

Molecular dynamics simulation study of AG10 and tafamidis binding to the Val122Ile transthyretin variant.

Molecular dynamics (MD) simulations were used to investigate the binding of four ligands to the Val122Ile mutant of the protein transthyretin. Dissociation, misfolding, and subsequent aggregation of mutated transthyretin proteins are associated with the disease Familial Amyloidal Cardiomyopathy. The ligands investigated were the drug candidate AG10 and its decarboxy and N-methyl derivatives along with the drug tafamidis. These ligands bound to the receptor in two halogen binding pockets (HBP) designated AB and A'B'. Inter-ligand distances, solvent accessible surface areas, root mean squared deviation measurements, and extracted structures showed very little change in the AG10 ligands' conformations or locations within the HBP during the MD simulation. In addition, the AG10 ligands experienced stable, two-point interactions with the protein by forming hydrogen bonds with Ser-117 residues in both the AB and A'B' binding pockets and Lysine-15 residues found near the surface of the receptor. Distance measurements showed these H-bonds formed simultaneously during the MD simulation. Removal of the AG10 carboxylate functional group to form decarboxy-AG10 disrupted this two-point interaction causing the ligand in the AB pocket to undergo a conformational change during the MD simulation. Likewise, addition of a methyl group to the AG10 hydrazone functional group also disrupted the two-point interaction by decreasing hydrogen bonding interactions with the receptor. Finally, MD simulations showed that the tafamidis ligands experienced fewer hydrogen bonding interactions than AG10 with the protein receptor. The tafamidis ligand in pocket A'B' was also found to move deeper into the HBP during the MD simulation.

Characterization of Amino Acid Based Molecular Micelles with Molecular Modeling.

The enantiomers of chiral drugs often have different potencies, toxicities, and biochemical properties. Therefore, the FDA and other worldwide regulatory agencies require manufactures to test and prove the enantiomeric purity of chiral drugs. Amino acid based molecular micelles (AABMM) have been used in chiral CE separations since the 1990's because of their low environmental impact and because their properties can easily be tuned by changing the amino acids in the chiral surfactant headgroups. Using molecular dynamics simulations to investigate the structures and properties of AABMM is part of an ongoing study focusing on investigating and elucidating the factors responsible for chiral recognition with AABMM. The results will be useful for the proper design and selection of more efficient chiral selectors. The micelles investigated contained approximately twenty covalently linked surfactant monomers. Each monomer was in turn composed of an undecyl hydrocarbon chain bound to a dipeptide headgroup containing of all combinations of L-Alanine, L-Valine, and L-Leucine. These materials are of interest because they are effective chiral selectors in capillary electrophoresis separations. Molecular dynamics simulation analyses were used to investigate how the sizes and positions of the headgroup amino acid R-groups affected the solvent accessible surface areas of each AABMM chiral center. In addition, headgroup dihedral angle analyses were used to investigate how amino acid R-group size and position affected the overall headgroup conformations. Finally, distance measurements were used to study the structural and conformational flexibilities of each AABMM headgroup. All analyses were performed in the context of a broader study focused on developing structure-based predictive tools to identify the factors responsible for (a) self-assembly, (b) function, (c) higher ordered structure and (d) molecular recognition of these amino acid based molecular micelles.

Investigation of Chiral Recognition by Molecular Micelles with Molecular Dynamics Simulations.

Molecular dynamics simulations were used to characterize the binding of the chiral drugs chlorthalidone and lorazepam to the molecular micelle poly-(sodium undecyl-(L)-leucine-valine). The project's goal was to characterize the nature of chiral recognition in capillary electrophoresis separations that use molecular micelles as the chiral selector. The shapes and charge distributions of the chiral molecules investigated, their orientations within the molecular micelle chiral binding pockets, and the formation of stereoselective intermolecular hydrogen bonds with the molecular micelle were all found to play key roles in determining where and how lorazepam and chlorthalidone enantiomers interacted with the molecular micelle.

Nuclear Magnetic Resonance Investigation of the Effect of pH on Micelle Formation by the Amino Acid-Based Surfactant Undecyl l-Phenylalaninate.

Micelle formation by the anionic amino acid-based surfactant undecyl l-phenylalaninate (und-Phe) was investigated as a function of pH in solutions containing either Na+, l-arginine, l-lysine, or l-ornithine counterions. In each mixture, the surfactant's critical micelle concentration (CMC) was the lowest at low pH and increased as solutions became more basic. Below pH 9, surfactant solutions containing l-arginine and l-lysine had lower CMC than the corresponding solutions with Na+ counterions. Nuclear magnetic resonance (NMR) diffusometry and dynamic light scattering studies revealed that und-Phe micelles with Na+ counterions had hydrodynamic radii of approximately 15 Å throughout the investigated pH range. Furthermore, l-arginine, l-lysine, and l-ornithine were found to bind most strongly to the micelles below pH 9 when the counterions were cationic. Above pH 9, the counterions became zwitterionic and dissociated from the micelle surface. In und-Phe/l-arginine solution, counterion dissociation was accompanied by a decrease in the hydrodynamic radius of the micelle. However, in experiments with l-lysine and l-ornithine, micelle radii remained the same at low pH when counterions were bound and at high pH when they were not. This result suggested that l-arginine is attached perpendicular to the micelle surface through its guanidinium functional group with the remainder of the molecule extending into solution. Contrastingly, l-lysine and l-ornithine likely bind parallel to the micelle surface with their two amine functional groups interacting with different surfactant monomers. This model was consistent with the results from two-dimensional ROESY (rotating frame Overhauser enhancement spectroscopy) NMR experiments. Two-dimensional NMR also showed that in und-Phe micelles, the aromatic rings on the phenylalanine headgroups were rotated toward the hydrocarbon core of micelle.

Molecular Dynamics Simulation and NMR Investigation of the Association of the ß-Blockers Atenolol and Propranolol with a Chiral Molecular Micelle.

Molecular dynamics simulations and NMR spectroscopy were used to compare the binding of two ß-blocker drugs to the chiral molecular micelle poly-(sodium undecyl-(L)-leucine-valine). The molecular micelle is used as a chiral selector in capillary electrophoresis. This study is part of a larger effort to understand the mechanism of chiral recognition in capillary electrophoresis by characterizing the molecular micelle binding of chiral compounds with different geometries and charges. Propranolol and atenolol were chosen because their structures are similar, but their chiral interactions with the molecular micelle are different. Molecular dynamics simulations showed both propranolol enantiomers inserted their aromatic rings into the molecular micelle core and that (S)-propranolol associated more strongly with the molecular micelle than (R)-propranolol. This difference was attributed to stronger molecular micelle hydrogen bonding interactions experienced by (S)-propranolol. Atenolol enantiomers were found to bind near the molecular micelle surface and to have similar molecular micelle binding free energies.

A Molecular Dynamics Simulation Study of the Association of 1,1'-Binaphthyl-2,2'-diyl hydrogenphosphate Enantiomers with a Chiral Molecular Micelle.

Molecular dynamics (MD) simulations were used to investigate the binding of 1,1'-binaphthyl-2,2'-diyl hydrogenphosphate (BNP) enantiomers to the molecular micelle poly-(sodium undecyl-(L,L)-leucine-valine) (poly(SULV)). Poly(SULV) is used as a chiral selector in capillary electrophoresis separations. Four poly(SULV) binding pockets were identified and either (R)-BNP or (S)-BNP were docked into each pocket. MD simulations were then used to identify the preferred BNP binding site. Within the preferred site, both enantiomers formed hydrogen bonds with poly(SULV) and penetrated into the poly(SULV) core. Comparisons of BNP enantiomer binding to the preferred poly(SULV) pocket showed that (S)-BNP formed stronger hydrogen bonds, moved deeper into the binding site, and had a lower poly(SULV) binding free energy than the (R) enantiomer. Finally, MD simulation results were in agreement with capillary electrophoresis and NMR experiments. Each technique showed (S)-BNP interacted more strongly with poly(SULV) than (R)-BNP and that the site of chiral recognition was near the poly(SULV) leucine chiral center.

A Molecular Dynamics Simulation Study of Two Dipeptide Based Molecular Micelles: Effect of Amino Acid Order.

Molecular dynamics (MD) simulations were used to compare the structures of the chiral molecular micelles (MM) poly-(sodium undecyl-(L,L)-leucine-valine) (poly(SULV)) and poly-(sodium undecyl-(L,L)-valine-leucine) (poly (SUVL)). Both MM contained polymerized surfactant monomers tenninated by chiral dipeptide headgroups. The study was undertaken to investigate why poly(SULV) is generally a better chiral selector compared to poly(SUVL) in electrokinetic chromatography separations. When comparing poly(SULV) to poly(SUVL), poly(SULV) had the more conformational flexible dipeptide headgroup and hydrogen bond analyses revealed that the poly(SULV) headgroup conformation allowed a larger number of intramolecular hydrogen bonds to form between monomer chains. In addition, a larger number of water molecules surrounded the chiral centers of the poly(SULV) molecular micelle. Poly(SULV) was also found to have a larger solvent accessible surface area (SASA) than poly(SUVL) and fluctuations in the poly(SULV) SASA during the MD simulation allowed dynamic monomer chain motions expected to be important in chiral recognition to be identified. Finally, approximately 50% of the Na+ counterions were found in the first three solvation shells surrounding both MM, with the remainder located in the bulk. Overall the MD simulations point to both greater headgroup flexibility and solvent and analyte access to the chiral centers of the dipeptide headgroup as factors contributing to the enhanced chiral selectivity observed with poly(SULV).

Investigation of Chiral Molecular Micelles by NMR Spectroscopy and Molecular Dynamics Simulation.

NMR spectroscopy and molecular dynamics (MD) simulation analyses of the chiral molecular micelles poly-(sodium undecyl-(L,L)-leucine-valine) (poly-SULV) and poly-(sodium undecyl-(L,L)- valine-leucine) (poly-(SUVL)) are reported. Both molecular micelles are used as chiral selectors in electrokinetic chromatography and each consists of covalently linked surfactant chains with chiral dipeptide headgroups. To provide experimental support for the structures from MD simulations, NOESY spectra were used to identify protons in close spatial proximity. Results from the NOESY analyses were then compared to radial distribution functions from MD simulations. In addition, the hydrodynamic radii of both molecular micelles were calculated from NMR-derived diffusion coefficients. Corresponding radii from the MD simulations were found to be in agreement with these experimental results. NMR diffusion experiments were also used to measure association constants for polar and non-polar binaphthyl analytes binding to both molecular micelles. Poly(SUVL) was found to bind the non-polar analyte enantiomers more strongly, while the more polar analyte enantiomers interacted more strongly with poly(SULV). MD simulations in tum showed that poly(SUL V) had a more open structure that gave greater access for water molecules to the dipeptide headgroup region.

NMR characterization of 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate binding to chiral molecular micelles.

NMR spectroscopy was used to characterize the binding of the chiral compound 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate (BNP) to five molecular micelles with chiral dipeptide headgroups. Molecular micelles have covalent linkages between the surfactant monomers and are used as chiral mobile phase modifiers in electrokinetic chromatography. Nuclear overhauser enhancement spectroscopy (NOESY) analyses of (S)-BNP:molecular micelle mixtures showed that in each solution the (S)-BNP interacted predominately with the N-terminal amino acid of the molecular micelle's dipeptide headgroup. NOESY spectra were also used to generate group binding maps for (S)-BNP:molecular micelle mixtures. In these maps, percentages are assigned to the (S)-BNP protons to represent the relative strengths of their interactions with a specified molecular micelle proton. All maps showed that (S)-BNP inserted into a previously reported chiral groove formed between the molecular micelle's dipeptide headgroup and hydrocarbon chain. In the resulting intermolecular complexes, the (S)-BNP protons nearest to the analyte phosphate group were found to point toward the N-terminal Halpha proton of the molecular micelle headgroup. Finally, pulsed field gradient NMR diffusion experiments were used to measure association constants for (R) and (S)-BNP binding to each molecular micelle. These K values were then used to calculate the differences in the enantiomers' free energies of binding, Delta(DeltaG). The NMR-derived Delta(DeltaG) values were found to scale linearly with electrokinetic chromatography (EKC) chiral selectivities from the literature.

Using perdeuterated surfactant micelles to resolve mixture components in diffusion-ordered NMR spectroscopy.

Diffusion-ordered NMR spectroscopy resolves mixture components on the basis of differences in their respective diffusion coefficients or molecular sizes. However, when components have near-identical diffusion coefficients, they are not resolved in the diffusion dimension of a diffusion-ordered spectroscopy (DOSY) spectrum. Adding surfactant micelles to these mixtures has been shown to enhance resolution when the component molecules interact differentially with the micelles. This approach is similar to that used in electrokinetic chromatography (EKC) where modifiers like micelles or polymers are used to enhance the separation of mixture components. In this study, perdeuterated surfactants are added to analyte mixtures studied with the DOSY technique. Since no micelle resonances appear in the mixture spectra, the difficulty associated with performing biexponential analyses in spectral regions where analyte and surfactant resonances overlap is avoided. The approach is demonstrated using mixtures of peptides with near-identical diffusion coefficients.

NMR characterization of the host-guest inclusion complex between beta-cyclodextrin and doxepin.

The interaction between doxepin, a member of the tricyclic antidepressant (TCA) class of drugs, with beta-cyclodextrin (beta-CD) was investigated using NMR. Several TCAs have been reported to form a complex with beta-CD having 1:1 stoichiometry. Previous results from UV-visible spectroscopy, fluorescence measurements, and molecular modeling indicated that for imipramine, desipramine, and amitriptyline, the TCA aliphatic tail is included in the cyclodextrin cavity with apparently no interaction of the tricyclic ring. An alternative view of the doxepin-beta-CD complex is presented in this work using analysis of complexation-induced chemical shifts (CICSs), the method of continuous variation (Job's analysis), and analysis of ROESY spectra. The Job's plot derived from the NMR spectral data confirms that the complex formed has 1:1 stoichiometry. The largest changes in the CICS data were observed for the aromatic protons of one of the doxepin rings, with much smaller chemical shift changes observed for the protons of the other aromatic ring and the doxepin tail. Perhaps the most significant evidence for inclusion of the doxepin tricyclic ring is the strong ROESY cross peaks between the doxepin aromatic resonances and the protons located inside the beta-CD cavity. Changes in the doxepin (1)H NMR spectrum and the behavior of ROESY exchange cross peaks suggest that inclusion complex formation decreases the rate of internal motions of doxepin.

Understanding chiral molecular micellar separations using steady-state fluorescence anisotropy, capillary electrophoresis, and NMR.

Chiral separations employing four diastereomers of poly sodium N-undecanoyl leucylvalinate (p-SULV) as chiral selectors are probed by use of MEKC, steady-state fluorescence anisotropy, and NMR. By employing diastereomers and thus altering the stereochemistry of a single amino acid in a systematic way, one may control the enantiorecognition ability of the chiral selector. As a result, one can gain a better understanding of the mechanisms of chiral recognition for the two classes of neutral or anionic chiral analytes studied. Evaluation of the chiral interactions leading to chiral separations confirmed our earlier observation of a strong relationship between the selectivity (alpha) observed using a chromatographic separation technique (MEKC) and that determined from the spectroscopic parameter, beta. A linear alpha versus beta relationship was observed for the molecular micelle p-(L)-SULV with all eight analytes included in this study. However, as we earlier predicted, different groups of analytes had different slopes, i.e., values of m, suggesting different chiral separation mechanisms. Evaluation of the data allowed a grouping of the analytes according to the primary site of chiral interaction with the leucine or valine moiety of molecular micelle chiral headgroup.

Use of NMR binding interaction mapping techniques to examine interactions of chiral molecules with molecular micelles.

NMR spectroscopy was used to investigate the association of four chiral molecules with the molecular micelle poly(sodium N-undecanoyl-l-leucylvalinate) (poly(SULV)). Adding poly(SULV) to the background electrolyte in electrokinetic chromatography (EKC) allows enantiomeric resolution to be achieved because enantiomers interact differentially with the chiral centers on the micelle headgroups as they both move in the electric field. Pulsed field gradient diffusion experiments were used to measure molecular micelle association constants for enantiomers of each analyte. These association constants were consistent with EKC elution order for the compounds 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate (BNP), 1,1'-bi-2-naphthol (BOH), and Troger's base. In addition, nuclear Overhauser enhancement spectroscopy, nuclear Overhauser effect difference, and intermolecular cross relaxation diffusion experiments were used to generate binding interaction maps for each chiral analyte. These maps showed that BNP and BOH inserted into the surfactant headgroup's major chiral groove and interacted predominately with the leucine chiral center. (+)-Troger's base was also found to insert into the major chiral groove. However, this compound instead interacted with the valine chiral atom. In diffusion experiments with long diffusion times, the linearized diffusion plots for each analyte-molecular micelle mixture showed curvature characteristic of intermolecular cross relaxation. The magnitude of this effect scaled linearly with the analytes' free energies of binding.

An improved method for suppressing protein background in PFG NMR experiments to determine ligand diffusion coefficients in the presence of receptor.

In NMR diffusion experiments to study ligand-protein binding equilibria, the spectral background due to broad protein resonances can contribute significantly to the measured ligand signal intensity resulting in erroneous binding affinities. One method to suppress the protein spectral background involves coupling a CPMG pulse train before or after the BPPSTE pulse sequence to allow for differential T(2) relaxation of the broad protein resonances. Here, we present an improved method, the Gradient Phase Encoded Spin-lock (GraPES) experiment that integrates the relaxation filter into the diffusion period. Compared with sequential CPMG-BPPSTE pulse sequences, GraPES offers effective suppression of the protein background with improved signal-to-noise ratios and shorter experiment times.

A pulsed field gradient NMR diffusion investigation of enkephalin peptide-sodium dodecyl sulfate micelle association.

Pulsed field gradient NMR (PFG-NMR) diffusion experiments were used to investigate the binding of leucine and methionine enkephalin peptides to anionic sodium dodecyl sulfate (SDS) micelles. The study was undertaken to investigate the mechanism of interaction between enkephalin peptides and SDS micelles and to determine if NMR-derived association constants, K(eq), can predict the elution order in electrokinetic chromatography (EKC). In EKC, peptides are separated on the basis of their interactions with micelles. The Leu-enkephalin peptide-micelle association constant increased from 130 +/- 8 to 1459 +/- 57 and 1744 +/- 64 M(-1), respectively, when an Arg or Lys was added to the C-terminus. The association constant of Leu-enkephalinamide was approximately equal to that of Leu-enkephalin-Arg. Substitution of Phe4 with a Trp or Gly2 with an Ala in the Leu-enkephalin peptides also increased the micelle binding affinity. These results confirm that the interaction of Leu-enkephalin peptides with SDS micelles is largely electrostatic and that the non-polar amino acid side chains interact with the hydrophobic micelle core. The peptide-micelle association constants for the cationic Met-enkephalin peptides were also greater than their zwitterionic counterparts. For example, the Met-enkephalin K(eq) value was 162 +/- 9 M(-1), while the association constants for Met-enkephalin-Arg, Met-enkephalin-Lys, and Met-enkephalinamide were, respectively, 674 +/- 31, 426 +/- 23, and 453 +/- 27 M(-1). In both Met-enkephalin and Met-enkephalinamide, replacing Gly2 with an Ala did not significantly increase the association constant. These results confirm that with the Met-enkephalin peptides, there was little or no interaction of the amino acid side chains with the micelle core. For both the Leu-enkephalin and Met-enkephalin peptides, the association constants were consistent with EKC results, in that the peptides with smaller K(eq) values were found to elute before those with larger association constants.

Slice thickness in the assessment of medial and lateral tibial cartilage volume and accuracy for the measurement of change in a longitudinal study.

OBJECTIVE: The optimal magnetic resonance image (MRI) slice thickness required to assess cartilage volume accurately and efficiently in cross-sectional and longitudinal studies is unknown. We compared cartilage volume measured from MRI of the knees using different slice thicknesses (1.5 to 7.5 mm) and assessed longitudinal change. METHODS: A total of 123 subjects with osteoarthritis had baseline and followup MRI on their symptomatic knee at 2 years. Medial and lateral tibial cartilage volumes were calculated using increasing slice thickness by extracting each second, third, fourth, or fifth slice area to calculate total volume, which was compared to the "gold standard" volume calculated from the original 1.5 mm slices. RESULTS: There was little difference in the average medial and lateral tibial cartilage volume observed as the slice thickness increased from 1.5 to 7.5 mm; medial tibial cartilage volume ranged from 1750 microl to 1787 microl and lateral tibial cartilage volume ranged from 1949 microl to 2007 microl. There was also little absolute difference in the average change in medial and lateral tibial cartilage volume measured over 2 years. However, with increasing slice thickness, there was a decreased correlation between the tibial cartilage volume change calculated from the increased slice thickness, with the lowest correlation being 0.77 (p < 0.001) when the lateral cartilage volume calculated from the 7.5 mm slice was compared to the 1.5 mm slices. CONCLUSION: Increasing slice thickness may provide sufficiently accurate measurement of tibial cartilage volume and change over time in some studies. This would result in reduction in MRI scanning and postimaging processing time, which has the potential of increasing the feasibility of this technique.

The interactions of the HIV gp41 fusion peptides with zwitterionic membrane mimics determined by NMR spectroscopy.

The wild-type (wt) N-terminal 23-residue fusion peptide (FP) of the human immunodeficiency virus (HIV) fusion protein gp41 and its V2E mutant have been studied by nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles as membrane mimics. A number of NMR techniques have been used. Pulsed field-gradient diffusion measurements in DPC and in 4:1 DPC/sodium dodecylsulfate mixed micelles showed that there is no major difference between the partition coefficients of the fusogenic wt peptide and the V2E mutant in these micelles, indicating that there is no correlation between the activity of the fusion peptides and their membrane affinities. The nuclear Overhauser enhancement (NOE) patterns and the chemical shift index for these two peptides indicated that both FP are in an alpha helical conformation between the Ile4 to Leu12 or to Ala15 region. Simulated annealing showed that the helical region extends from Ile4 to Met19. The two FPs share similar conformational characteristics, indicating that the conformation of the FP is not an important factor determining its activity. The spin-label studies, utilizing spin labels 5- and 16-doxystearic acids in the DPC micelles, provided clear indication that the wt FP inserts its N-terminus into the micelles while the V2E mutant does not insert into the micelles. The conclusion from the spin-label results is corroborated by deuterium amide proton exchange experiments. The correlation between the oblique insertion of the FP and its fusogenic activity is in excellent agreement with results from our molecular dynamics simulation and from other previous studies.