The conformation of xanthene dyes in the myosin sulfhydryl one binding site Part I. Methods and model systems

Derivatives of the fluorescent probes fluorescein and rhodamine specifically and covalently modify the highly reactive thiol (SH1) of myosin subfragment 1 (S1). Both probes develop circular dichroism (CD) upon modification of SH1 at the visible absorption band of the chromophore. A model system of chiral complexing agents (aromatic chiral amines) interacting with fluorescein in solvent develops a CD signal that mimics that produced by S1. The model system suggests that a specific interaction of the probe with an aromatic chiral residue in the SH1 binding pocket induces the CD signal. Several other spectroscopic signals, including absorption and fluorescence intensity and anisotropy, characterize the fluorescein or rhodamine binding to SH1. A coupled dipole method is adapted to interpret these spectroscopic signals in terms of the probe-S1 complex conformation. The computation of the orientation of the principal hydrodynamic frame (PHF) of S1 from its crystallographic -carbon backbone structure permits the known orientation of the probe in the PHF of S1 to further constrain the conformation of the probe-S1 complex. The coupled dipole interpretation of spectroscopic data combined with constraints relating the probe dipole orientation to the PHF of S1 determines the conformation of the probe-S1 complex. The methods developed here are applied to the spectroscopic signals from fluorescein or rhodamine in the SH1 binding site of S1 to obtain an atomic resolution model of the probe-S1 conformation [Ajtai and Burghardt, Biochemistry, 34 (1995) 15943–15952].     

The conformation of peptide thymosin α1 in solution and in a membrane-like environment by circular dichroism and NMR spectroscopy. a possible model for its interaction with the lymphocyte membrane

The 28-residue peptide thymosin α1 was studied by circular dichroism and two-dimensional NMR. Circular dichroism indicates that thymosin α1 in water solution does not assume a preferred conformation, while in the presence of small unilamellar vesicles of dimiristoylphosphatidylcholine and dimiristoylphosphatidic acid (10:1) and in sodium dodecyl sulphate, it assumes a partly structured conformation. Presence of zinc ions produces similar effects. In a more hydrophobic environment like a solution of a mixed solvent water-2,2,2 trifluoroethanol, it adopts a structured conformation. NMR spectra indicated that in this mixture as solvent, thymosin α1 has a structure characterized by two regions. A β-turn is present between residue 5 and residue 8, while the region between residues 17 and 24 shows an α helix conformation. These changes of conformation in different environments may be considered structural requirements in the steps of its interaction with the lymphocyte membrane. In fact, these conformational changes may correspond to the first event of the mechanism of lymphocyte activation in the immune response modulation by thymosin α1.     

Determination of absolute configurations by X-ray crystallography and 1H NMR anisotropy

To determine the absolute configurations of chiral compounds, many spectroscopic and diffraction methods have been developed. Among them, X-ray crystallographic Bijvoet method, CD exciton chirality method, and the combination of vibrational circular dichroism and quantum mechanical calculations are of nonempirical nature. On the other hand, X-ray crystallography using a chiral internal reference, and 1H NMR spectroscopy using chiral anisotropy reagents are relative and/or empirical methods. In addition to absolute configurational determinations, preparations of enantiopure compounds are strongly desired. As chiral reagents useful for both the preparation of enantiopure compounds by HPLC separation and the simultaneous determination of their absolute configurations, we have developed camphorsultam dichlorophthalic acid (CSDP acid) for X-ray crystallography and 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid) for 1H NMR spectroscopy. In this review, the principles and applications of these X-ray and NMR methods are explained using mostly our own data.     

Chiral Sulfur Compounds Studied by Raman Optical Activity: tert-Butanesulfinamide and its Precursor tert-Butyl tert-Butanethiosulfinate

Two chiral sulfur compounds, tert-butyl tert-butanethiosulfinate (1) and tert-butanesulfinamide (2), with inversion of configuration, have been studied by Raman optical activity (ROA) and electronic circular dichroism combined with density functional theory calculation. With the S-S linkage in 1, the couplings between the two tertiary carbon atoms often generate large ROA signals, whereas the tertiary carbon atom itself generally makes a large contribution to ROA signals in 2 for similar vibrational modes. The conformational dependence of ROA parameters provides probing conformation around the S-S bond from a new perspective. The simultaneous use of electronic circular dichroism and ROA is warranted to extract reliable conformational information. ROA provides a suitable candidate for the stereochemical study of chiral sulfur compounds, especially its capability of sensing the conformation around the S-S bond. Chirality 24:731-740, 2012. ? 2012 Wiley Periodicals, Inc.     

Chiral hplc-cd studies of the antituberculosis drug (+)-ethambutol

Long standing errors in major pharmacopoeiae (BP,USP, and Eu.Ph.) concerning the absolute stereochemistry of the widely used antituberculosis drug (+)-ethambutol have been clarified by unambiguous synthesis and chiral HPLC. on a Pirkle, covalent D -phenylglycine column of perbenzoyl derivatives of each stereomer; the enantiomeric (?)-(R,R) and (+)-(S,S)-ethambutols together with the optically inactive (meso)-(R,S)-ethambutol. This paper describes how circular dichroism (CD) alone and combined with HPLC is used to demonstrate this chiral separation and also to confirm the absolute stereochemistry of each stereomer of ethambutol and its synthetic precursor 2-aminobutan-1-ol from studies of ?exciton coupling.”? The strengths and weaknesses of these chiral techniques are discussed.     

Separation and aquatic toxicity of enantiomers of 1-(substituted phenoxyacetoxy)alkylphosphonate herbicides

A series of organophosphorous compounds (OPs), 1-(substituted phenoxyacetoxy)alkylphosphonates containing a chiral carbon atom, show notable herbicidal activities. In this study, the enantioselective separation and biological toxicity of all these compounds were investigated. The enantioselective separation on the columns of Chiralpak AD, Chiralpak AS, Chiralcel OD, and Chiralcel OJ were compared under various chromatographic conditions. All the analytes investigated obtained baseline resolution (R(s) > 1.5) on Chiralpak AD column, which showed best chiral separation capacity. Further investigation was carried out on Chiralpak AD to evaluate the influence of the mobile phase composition and column temperature. The effect of the structural features on discrimination was also examined. The resolved enantiomers were distinguished by their signs of circular dichroism. The acute aquatic toxicity of enantiomers and racemate to Daphnia magna (D. magna) were assessed. The in vivo assays showed that compound 3 was about 2-148.5 times more toxic than the other four analogues to D. magna. The racemates of compounds 3 and 5 showed intermediate toxicity compare to their enantiomers, while those of compounds 1, 2, and 4 showed synergistic or antagonistic effect. These results suggest that the biological toxicity of chiral OPs to nontarget organisms is enantioselective and therefore should be evaluated with their pure enantiomers.     

Specific optical rotation is a versatile tool for the identification of critical micelle concentration and micellar growth of tartaric acid–based diastereomeric amphiphiles

Four novel tartaric acid–based diastereomeric chiral amphiphiles, two being enantiomers of the other two, have been synthesized and investigated using chiroptical spectroscopic methods, along with tensiometry and dynamic light scattering experiments. We found that an inflection point in specific optical rotation (SOR) values at ~0.32 mM corresponds to the critical micelle concentration (CMC). The increase in magnitude of SOR values beyond CMC corresponds to the growth of aggregates. For enantiomers, oppositely signed SOR values were observed, ruling out the possibility for the presence of aggregation size mediated artefacts. SOR values did not exhibit concentration dependence for a chiral tartaric acid based non‐aggregating analogue further establishing the absence of artefacts or anomalous interaction of tartaric acid based head group with solvent. Electronic circular dichroism spectra showed no significant changes in band positions or intensities with concentration. Due to the requirement for higher concentrations (~200 mM) needed to obtain vibrational circular dichroism spectra, these measurements are not found to be useful for studying concentration dependent properties of chiral amphiphiles.     

Enzymatic resolution of racemic amines in a continuous reactor in organic solvents

An enzymatic process has been developed for the continuous production of the pharmaceutically important intermediate (R)-1-aminoindan and of the chiral resolving agent (R)-1-(1-naphthyl)ethylamine. The process consists of the subtilisin catalyzed stereoselective aminolysis of the racemic primary amine with an active ester in organic solvent. The competing nonenzymatic reaction has been suppressed by appropriate choice of solvent and reactant's concentration and by minimizing the time of contact between the amine and the active ester. Subtilisin was immobilized on glass beads and the reaction carried out in a continuous-flow column bioreactor. By using a 450-mL column bioreactor containing 5.7 g of subtilisin immobilized on 570 g of glass beads, 1.6 kg of racemic 1-(1-naphthyl)ethylamine was resolved after 320 h of continuous operation with only a slight loss of the enzymatic activity. During the whole process, the optical purity of the chiral amine eluting from the column was higher than 90%. A facile procedure was developed for separating the unreacted (R)-amine from the (S)-amide and for the recycling of the solvent 3-methyl-3-pentanol and the active ester 2,2,2-trifluoroethyl butyrate. (c) 1992 John Wiley & Sons, Inc.     

Enantiomeric separation of imidazolinone herbicides using chiral high-performance liquid chromatography

Chiral high-performance liquid chromatography (HPLC) is one of the most powerful tools to prepare enantiopure standards of chiral compounds. In this study, the enantiomeric separation of imidazolinone herbicides, i.e., imazethapyr, imazapyr, and imazaquin, was investigated using chiral HPLC. The enantioselectivity of Chiralpak AS, Chiralpak AD, Chiralcel OD, and Chiralcel OJ columns for the three analytes was compared under similar chromatographic conditions. Chiralcel OJ column showed the best chiral resolving capacity among the test columns. The resolved enantiomers were distinguished by their signs of circular dichroism detected at 275 nm and their structures confirmed with LC-mass spectrometric analysis. Factors affecting the chiral separation of imidazolinones on Chiralcel OJ column were characterized. Ethanol acted as a better polar modifier than the other alcohols including 2-propanol, 1-butanol, and 1-pentanol. Although the acidic modifier in the mobile phase did not influence chiral recognition, it was necessary for reducing the retention time of enantiomers and suppressing their peak tailing. Thermodynamic evaluation suggests that enantiomeric separation of imidazolinones on Chiralcel OJ column is an enthalpy-driven process from 10 to 40 degrees C. This study also shows that small amounts of pure enantiomers of imidazolinones may be obtained by using the analytical chiral HPLC approach.     

Determination of the enantiomeric purity of the phytoalexins spirobrassinins by 1H NMR using chiral solvation

A simple and inexpensive method for enantiomeric discrimination of the phytoalexins spirobrassinin (1), 1-methoxyspirobrassinin (2) and synthetic analog 1-methylspirobrassinin (6) using the chiral solvating agent 2,2,2-trifluoro-1-(9-anthryl)ethanol in C(6)D(6) is described. Using this method the enantiomeric composition of each sample can be determined accurately by (1)H NMR and the compounds can be recovered readily by chromatography.     

Hints of nonhierarchical folding of acidic fibroblast growth factor

We have analyzed by circular dichroism (CD) and proton nuclear magnetic resonance (NMR) the helical propensity of the all-beta protein acidic fibroblast growth factor (aFGF) and two peptides corresponding to beta-strand 8 (beta8 peptide, amino acids 95-107) and the beta-strand 8/turn/beta-strand 9 hairpin (beta8/9 peptide, amino acids 95-114), which has been involved in receptor binding. A secondary structure prediction of aFGF carried out by several procedures labels the 95-104 sequence as predominantly alpha-helical. A titration of aFGF with 2,2,2-trifluoroethanol (TFE) induces a change in the far-UV CD spectrum of the protein giving rise to a prominent alpha-helical shape (22% alpha-helix). The cooperativity of the transition and the moderate TFE concentrations used (midpoint at 24%) suggest that the effect of TFE is specific. Moreover, a titration performed at pH 2 yields a higher amount of alpha-helix (55%) at a smaller TFE concentration. Synthetic peptides containing the beta8 and beta8/9 sequences display a random coil conformation at pH 7 but acquire alpha-helical structure in the presence of TFE, methanol, and SDS micelles. At pH below 3.0 a significant amount (20-30%) of alpha-helical conformation is present in both the beta8 and beta8/9 peptides even in the absence of other solvent additives. The secondary structure of the peptides was determined by proton nuclear magnetic resonance (1H NMR). These results suggest that the 95-114 sequence of aFGF has helical propensity and that the protein may fold nonhierarchically in the early steps of folding, acquiring its final beta-structure by a later interaction with the rest of the polypeptide.     

Enantioselective chromatography and absolute configuration of N,N-dimethyl-3-(naphthalen-2-yl)-butan-1-amines: potential sigma1 ligands

We describe the preparation of racemic N,N-dimethyl-3-(naphthalen-2-yl)-butan-1-amines, potential sigma1 ligands, and their resolution via chiral HPLC. In order to obtain enantiopure compounds, direct chromatographic methods of separation using chiral stationary phases were investigated. Different methods suitable for both analytical and semipreparative purposes are proposed. The best resolutions were achieved using cellulose tris (3,5-dimethylphenyl carbamate) (Chiralcel OD and OD-H) and amylose tris (3,5-dimethylphenyl carbamate) (Chiralpak AD). On the basis of the preliminary chromatographic results, the resolution of compound 1 was transferred onto a Chiralcel OD semipreparative column. The enantiomers were obtained in high enantiomeric excess. The configurational assignment was performed by circular dichroism. Computational analysis was used to explore the enantioselective recognition process of compound 1 with the Chiralcel OD stationary phase.     

Studies on tetrahydrofuranyl-5-fluorouracils. 2. NMR studies of 1-(tetrahydro-2-furanyl)-, 3-(tetrahydro-2-furanyl)-, and 1,3-bis(tetrahydro-2-furanyl)-5-fluorouracils.

Measurements of the 1H NMR spectra of the diastereoisomers of 1-(tetrahydro-2-furanyl)-5-fluorouracil, 3-(tetrahydro-2-furanyl)-5-fluorouracil, and 1,3-bis(tetrahydro-2-furanyl)-5-fluorouracil in the presence of tris[3-(2,2,2-trifluoro-1-hydroxyethylidene)-d-camphorato]europium(Eu(TFC)3) as a chiral shift reagent showed differences between the isomers in the chemical shift changes of the protons of C2'-H and C6-H etc.     

HPLC and NMR methods for the quantitation of the (R)-enantiomer in (−)-(S)-timolol maleate drug raw materials

HPLC and ¹H-NMR methods for the quantitation of the (R)-enantiomer in (?)-(S)-timolol maleate were developed and validated. The HPLC method requires a 25 cm × 4.6 mm 5 μm Chiracel OD-H (cellulose tris-3,5-dimethylphenylcarbamate) column, a mobile phase of 0.2% (v/v) diethylamine and 4% (v/v) isopropanol in hexane at a flow rate of 1 ml/min and UV detection at 297 nm. A system suitability test was devised to verify the separation of the (R)- and (S)-enantiomers of timolol from other drug-related impurities. The NMR method requires the use of a high-field NMR spectrometer (>360 MHz) and a chiral solvating agent, (?)-(R)-2,2,2-trifluoro-1-(9-anthrylethanol) (R-TFAE). The limits of quantitation were 0.05% and 0.2% (m/m) for HPLC and NMR, respectively. The methods were applied to the determination of the (R)-enantiomer in eight lots of raw material. The results for the two methods were in very good agreement, with results ranging from 0.1 to 4.1% (m/m) by HPLC and none detected to 4.3% (m/m) by NMR. The USP method for specific rotation was found to be unsuitable for detecting the presence of low levels of the (R)-enantiomer in (?)-(S)-timolol maleate. © 1994 Wiley-Liss, Inc.     

Conformational studies on chiral rhodium complexes by ECD and VCD spectroscopy

This article reports vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopic studies in acetonitrile on the chiral Rh(2)(O-Phe-Cbz)(1)(OAc)(3) and Rh(2)(O-Phe-Ac)(1)(OAc)(3) complexes (abbreviated Rh(2)Z(1) and Rh(2)Ac(1) , respectively; Phe, L-phenylalanine; Cbz, benzyloxycarbonyl; Ac, acetyl) supported by theoretical calculations. The ECD spectra of the complexes depend on temperature that indicates the conformational mobility of the chiral ligands. Calculations of the VCD spectra were performed at ab initio (DFT) level of theory using Gaussian 03 [B3LYP functional combined with the LANL2DZ basis set for the dirhodium core and the 6-31G(d) basis set for other atoms]. The population-weighted sums of the computed VCD spectra of the conformers are in excellent agreement with the experimental VCD spectra. The combination of the VCD and ECD spectroscopic methods led us to the structural characterization of the complexes.     

Circular dichroism and absorption study of the structure of methionine-enkephalin in solution

The circular dichroism and absorption spectra of methionine-enkephalin have been measured in aqueous solution, as functions of temperature and pH, and in 2,2,2-trifluoroethanol. Ranges covered were: 190–330 nm; 5–50°C; pH = 1–12. Absorption data provide no evidence for a strong intramolecular hydrogen bond involving the hydroxyl proton of the tyrosine residue. All data can be interpreted without assuming methionine-enkephalin preferentially occupies a single conformation when in dilute solution. The biologically important conformation is presumably one of many which are present to significant extent.     

Metabolites produced by the phytopathogenic fungus Rhizoctonia solani: isolation, chemical structure determination, syntheses and bioactivity

The isolation, structure determination, syntheses and biological activity of Nb-acetyltryptamine and three proline containing dioxopiperazines, cyclo(S-Pro-S-Leu) (2), cyclo(S-Pro-S-Ile) (3), and cyclo(S-Pro-S-Val) (4), from cultures of Rhizoctonia solani Kuhn are reported here for the first time. Despite the small amounts isolated, the absolute stereochemistry of these naturally occurring dioxopiperazines was established by 1H NMR using for the first time the chiral solvating agent (R)-(-)-2,2,2-trifluoro-1-(9-anthryl)ethanol.     

Solvent effects on the structure of rabbit Clq, a subcomponent of the first component of complement.

The effect of solvent conditions on the conformation of rabbit Clq was studied by both spectroscopic and nonspectroscopic methods. The conformation of Clq in buffered saline solutions at pH 7.4 or 6.0 did not differ significantly from Clq at twice the saline concentration as determined with circular dichroism, difference spectroscopy, and tritium-hydrogen exchange techniques. Addition of calcium to the buffers had no structural effects in any of the conditions examined. Hydrogen exchange experiments performed at pH 7.4 were also unaffected by magnesium, manganese, or ethylenediaminetetraacetic acid. With all the methods used a pH effect was observable between 5.1 and 8.3. From solvent perturbation difference spectroscopy results it was calculated that the equivalent of 10 +/- 2 and 6 +/- 1 mol of tyrosine and tryptophan/mol of Clq, respectively, became exposed at the lower pH. A small positive CD band in the 231 to 235 nm region decreased in wavelength and increased in magnitude as a function of decreasing pH, indicating tyrosine exposure at the lower pH and possibly changes in the collagen-like structure of Clq. Hydrogen exchange experiments indicate a small, but significant, conformation transition occurring in the pH 5 region and a stabilization of conformation between pH 6 to 8. From these results the conformational pH dependence was interpreted as an acid expansion of Clq with a minor conformational transition occurring between pH 5 AND 6. These effects may in part be associated with decreased Clq-Ig interactions which have been observed at the lower pH.     

The conformation of calmodulin: a substantial environmentally sensitive helical transition in Ca4-calmodulin with potential mechanistic function

The conformation of Ca4-calmodulin in solution, as assessed by far-UV peptide circular dichroism, contains significantly less alpha-helix than the proposed X-ray crystal structure. We now show that Ca4-calmodulin adopts significant additional helical structure in solution in the presence of a helicogenic solvent (50%, v/v, aqueous 2,2,2-trifluoroethanol or 50%, v/v, methylpentane-5,5-diol). We suggest that the long continuous helix (residues 66-92 of the crystal structure) is not necessarily a normal feature of the calmodulin structure in solution, and may be due in part to the conditions of crystallisation. This result is supported by time-resolved tyrosine fluorescence anisotropy studies indicating that Ca4-calmodulin in solution is an essentially compact globular structure which undergoes isotropic rotational motion. We conclude that, under appropriate ionic and apolar environmental conditions, Ca4-calmodulin undergoes a substantial helical transition, which may involve residues in the central region of the molecule. Such a transition could have an important function in determining specificity and affinity in interactions of calmodulin with different target sequences of Ca2+-dependent regulatory enzymes.     

Electronic and vibrational signatures of peptide helical structures: A tribute to Anton Mario Tamburro

Our efforts on the synthesis of peptides with well-characterized secondary structures, combined with detailed spectroscopic investigations, most of them performed in collaboration with internationally recognized experts, have allowed us to publish the electronic (electronic circular dichroism) and vibrational (FTIR absorption, vibrational circular dichroism, Raman, and Raman optical activity) signatures of the poorly studied peptide 3(10)-helix (and the related β-bend ribbon spiral conformation as well) in comparison with those already known for the classical α-helix.