Novel approach to determining the absolute configurations at the C3-positions of various types of sterols based on an induced circular dichroism

Circular dichroism (CD) spectra of the 2,2'-binaphthyl ester derived from Δ(5)-sterols showed not bisignate CD but diagnostic CD bands at around 210 and 240nm. These bands might be attributable to an interaction between an olefinic chromophore and a binaphthyl one. Various types of unsaturated sterols were thus derivatized followed by complete hydrogenation, to give saturated sterols. As a result, CD spectra of the binaphthyl derivatives of the saturated sterols showed bisignate curves centered at 240nm (3S(β): positive chirality; 3R(α): negative one). This suggested a straightforward and practical method for discriminating the absolute stereogenic center at the C-3 positions of sterols based on an induced CD. This finding should contribute significantly to the analysis of metabolites of various types of sterols.     

Circular dichroism of the nucleic acid monomers.

Circular dichroism spectra of the nucleic acid monomers have been measured in aqueous solution and extended into the vacuum ultraviolet region to about 166 nm. Measurements were made on ribo and deoxyribo derivatives of adenine, guanine, hypoxanthine, cytosine, thymine, and uracil derivatives both with and without the 5′-phosphate (with the exception of ribosyl thymine 5′-phosphate). Absorption spectra of the deoxyribonucleotides measured to about 175 nm are also presented. The results demonstrate that both the circular dichroism and absorption spectra observed below 200 nm are no more complicated than the spectra normally recorded above 200 nm. In most cases, the circular dichroism spectra of the various derivatives of a given base are similar, indicating that the conformations are similar. On the other hand, the differences among the circular dichroism spectra of the various derivatives of a given base are sufficient to identify a particular derivative. The average circular dichroism for the deoxyribonucleotides is compared with the circular dichroism of native E. coli DNA. The comparison reveals that the circular dichroism of DNA below 200 nm is due principally to the interaction between the bases rather than the intrinsic circular dichroism of the monomers. The monomer transitions are discussed in relationship to the absorption and circular dichroism spectra presented.     

The absolute configuration of 1-carboxyethyl substituents on common hexoses by circular dichroism

Determination of the absolute configuration of the 1-carboxyethyl substituent on a monosaccharide by circular dichroism measurements was found to be a sensitive and simple method. It relies on comparison of the spectrum of a 1-carboxyethyl substituted sugar or sugar derivative with the spectra of (R)- and (S)-lactic acid in the region 200-260 nm in which the (R)- and (S)-configuration give negative and positive deltaepsilon, respectively. The oligo- or poly-saccharide containing a 1-carboxyethyl substituted sugar is hydrolyzed to monomers and the 1-carboxyethyl substituted sugar isolated by chromatography. The CD spectrum obtained for the 1-carboxyethyl substituted sugar in water solution at pH 2 is then compared with spectra of (R)- and (S)-lactic acid. The sign for the absorption and a maximum of comparable intensity and appearance around 210 nm, identify the stereochemistry.     

Allosteric transitions in cobalt hemoglobins.

Circular dichroism and difference ultraviolet visible spectra were obtained for cobalt hemoglobin derivatives. At 287 nm the ellipticity difference between the oxy- and deoxycobaltohemoglobin is about one-half as great as that for the native proteins indicating smaller quaternary conformational changes for the former. Deoxygenation increases the Soret rotational strengths of both iron and cobalt hemoglobins to comparable degrees suggesting similar conformational changes for their aromatic residues near the "heme." Deoxygenation causes a much larger decrease of L band ellipticity for iron than cobalt hemoglobin. Circular dichroism spectra of nitrosylcobaltohemoglobin indicate the molecule to have a T quaternary structure. The circular dichroism spectra of cobaltihemoglobin do not seem to fit the patterns of the other cobalt derivatives and its 287 nm ellipticity is pH-dependent. From the shape of the Soret circular dichroism spectra, it is estimated that the transition dipole makes an angle with the line joining the two opposing pyrrole nitrogens of about 60 degrees for oxy- and deoxycobaltohemoglobin, 80 degrees for cobaltihemoglobin, as compared to 70 degrees for the native oxy- and deoxyhemoglobins. Inositol hexaphosphate has little or no effect on the circular dichroism spectra of cobalt hemoglobins in the 287 nm region, but it significantly increases the Soret rotational strength and decreases the L band ellipticity. The results are interpreted to mean that polyphosphates modify primarily the protein structure of hemoglobins at the tertiary level, and that the intersubunit interactions are weak in cobalt hemoglobins.     

Optical rotatory dispersion and circular dichroism of benzoyl polysaccharides

Measurements of optical rotatory dispersion (ORD) and circular dichroism (CD) were made in the range of 400–205 nm for polysaccharide tribenzoates such as 2,3,6-tri-O-benzoyl amylose (I), 2,3,4-tri-O-benzoyl dextran (II), tri-O-benzoyl pullulan (III), 2,3,6-tri-O-benzoyl cellulose (IV), 2,3,6-tri-O-benzoyl mannan (V), and polyglycan dibenzoates such as 2,3,-di-O-benzoyl amylose (VI), cellulose (VII), and mannan (VIII). All compounds exhibit Cotton effects in the region of their UV absorption bands (206–285 nm). Comparison of the corresponding di- and tribenzoyl polysaccharides shows a qualitative agreement in number, position and sign of the CD bands but differences in ellipticity magnitude. The disubstituted derivatives exhibit smaller amplitudes than the trisubstituted ones. The contribution of the C(6) chromophore (linked by a CH₂-group to the asymmetric C(5) atom) was determined to be of the same sign as the combined contribution of the C(2) and C(3) substituents. The CD bonds of the individual polysaccharide derivatives, which differ in number, sign, and position, were discussed in terms of the steric position of the single chromophores and the steric arrangement and interaction caused by the configuration of the polysaccharides. The optical behavior of these polysaccharide derivatives was found to be not strongly influenced by a definite chain conformation in solution.     

Vibrational Circular Dichroism (VCD) Reveals Subtle Conformational Aspects and Intermolecular Interactions in the Carnitine Family

Vibrational circular dichroism spectra (VCD) in the mid‐IR region and electronic circular dichroism (ECD) spectra for three carnitine derivatives in the form of hydrochloride salts were recorded in deuterated methanol solutions. Density Functional Theory calculations help one to understand the significance of the observed VCD bands. VCD and ECD spectra are informative about the absolute configuration of the molecule, but VCD data reveal also some conformational aspects in the N,N,N‐trimethyl moiety and inform us about intermolecular interactions gained from the carbonyl stretching region for the acyl substituted carnitines. Chirality 27:907–913, 2015. © 2015 Wiley Periodicals, Inc.     

The circular dichroism of lysozyme.

The circular dichroism spectra of hen egg white lysozyme, and of lysozyme derivatives in which tryptophan residues 62 or 108, or both, are selectively oxidized, have been measured as a function of pH over the range of 200 to 310 nm. Neither Trp-62 nor Trp-108 is principally responsible for the positive rotational strength in the 280 to 300 nm region. The spectrum in the 200 to 230 nm region is nearly the same in the native protein and in the derivatives, and is little affected by binding of saccharide. These results are used to reinterpret the circular dichroism spectra of the lysozymes and alpha-lactalbumins.     

Chirality and circular dichroism of oriented molecules and anisotropic phases

The results of the "Chirality measurement" circular dichroism along different viewing directions within molecules and phases-the anisotropy of circular dichroism can give suitable information in order to check helicity rules or to analyze the suprastructural chirality of films of organic materials, respectively. These results of the ACD spectroscopy (the CD of anisotropic phases and oriented molecules) show that in an oriented state different information about "chirality" will be gained from different viewing directions. ACD measurements of alpha,beta-unsaturated ketosteroids, TADDOLes and binaphthol derivatives were analyzed. The chiral induction of cholesteric phases, the helical twisting power, is introduced as another chirality measurement which may possibly be a new relative method for the determination of the absolute configuration. Copyright 2000 Wiley-Liss, Inc.     

Central-to-axial chirality transfer revealed by liquid crystals: a combined experimental and computational approach for the determination of absolute configuration of carboxylic acids with an α chirality centre

The conversion into 6,7-dihydro-5H-dibenz[c,e]azepine (DAZ) N-protected amides is a viable route for the determination of the absolute configuration of chiral 2-substituted carboxylic acids. The biphenyl moiety of DAZ, besides being a probe of chirality for the electronic circular dichroism (ECD) spectroscopy, makes these systems suitable for configuration assignment by exploiting the chirality amplification which occurs in nematic liquid crystals. To assess the reliability of the liquid crystal method in detecting the absolute stereochemistry of chiral amides bound to a biphenyl group, we measured the helical twisting power of a series of DAZ-N-protected amides and compared these data with the results obtained from ECD measurements. We will show that the liquid crystal method, corroborated by HTP predictions, is trustworthy with our biphenyl derivatives, even when ECD spectra are ambiguous for the presence of aryl moieties displaying strong UV absorptions in the same range of the biphenyl chromophore.     

Stereoselective metabolism of 7-methylbenz[a]anthracene: absolute configuration of five dihydrodiol metabolites and the effect of dihydrodiol conformation on circular dichroism spectra

7-Methylbenz[a]anthracene (7-MBA) was metabolized stereoselectively by rat liver microsomes to form five optically active dihydrodiols as the predominant metabolites. The dihydrodiols were purified by a combination of reversed-phase and normal-phase high performance liquid chromatography (HPLC). By comparison of their circular dichroism (CD) spectra with the corresponding benz[a]anthracene (BA) dihydrodiols of known absolute stereochemistry, the major dihydrodiol enantiomers of 7-MBA have been determined to have 1R,2R-, 3R,4R- and 10R , 11R - absolute configurations, respectively. Due to their quasi- diaxial conformations, the absolute configuration of trans-5,6- and trans-8,9-dihydrodiols, the two most abundant metabolites of 7-MBA, could not be determined by simple comparisons of their circular dichroism spectra with those of the quasidi -equatorial BA 5R, 6R - and 8R , 9R -dihydrodiols. The major enantiomers of the quasi- diaxial trans-5,6- and trans-8,9-dihydrodiol metabolites of 7-MBA were determined by comparison to the CD spectrum of 7-bromo-BA 5R, 6R -dihydrodiol and by the exciton chirality method to have R,R absolute stereochemistry. This study also revealed that the circular dichroism Cotton effects of an enantiomeric dihydrodiol of polycyclic aromatic hydrocarbons can be drastically altered if the conformation (quasi- diaxial vs. quasi di-equatorial ) of the dihydrodiol is changed.     

On the various types of circular dichroism induced on acridine orange bound to poly(S-carboxymethyl-L-cysteine).

Circular dichroism and absorption spectra are measured on mixed solutions of acridine orange and poly(S-carboxymethyl-L -cysteine) at different pH and P/D mixing ratios. The observed circular dichroism spectra are classified into several types, mainly based on the number and sign of circular dichroic bands in the visible region. Three of them are associated with the absorption spectra characteristic of dimeric dye or higher aggregates of dye. Type I is observed with solutions, of which the pH is acid and P/D is higher than 4, and it has an unsymmetrical pair of positive and negative dichroic bands at 470 and 430 nm. This type is induced on the dye bound to the polymer in the β-conformation. Types II and III are considered to be characteristic of randomly coiled polymers. Type II is exhibited by solutions of P/D higher than 1 at pH 5–7 and has two dichroic bands around the same wavelengths as Type I but with opposite signs and an additional positive band at 560 nm. Type III, shown by solutions of P/D 2–0.6 at pH 6–10.5, has three dichroic bands around the same wavelengths as Type II but with signs opposite to it. The other two types of circular dichroism, induced for the solutions of P/D less than 1 at slightly acid pH, are associated with the absorption spectra of monomeric dye and are observed with disordered or randomly coiled polymer. They have a pair of dichroic bands at 540 and 425 nm, and the signs of these bands are opposite to each other in these two types.     

Circular dichroism of soybean leghemoglobin.

Circular dichroic (CD) spectra of soybean leghemoglobin, and some of its liganded derivatives were measured over the wavelength range of 650 to 200 nm. The heme-related circular dichroic bands in the visible, Soret and ultraviolet wavelength regions exhibit Cotton effects characteristic of each of the compounds examined. The positions of the dichroic bands vary with ligand substitutions and the oxidation state of the iron. All leghemoglobin derivatives, except the apoprotein, exhibit negative circular dichroic bands in the region of Soret absorption. In this region the optical activity of compounds with high-spin moments is greater than that of compounds with low or intermediate spin moments. The ellipticity of the heme band at about 260 nm is also altered by ligand binding and spin state. The dichroic spectra in the far-ultraviolet region indicated a high extent of alpha-helical structure (about 70%) in the native leghemoglobin and its liganded derivatives. The helicality of the apoprotein seems to diminish suggesting a decrease caused by the removal of the heme.     

Circular dichroism of collagen, gelatin, and poly(proline) II in the vacuum ultraviolet.

Circular dichroism spectra for acid-soluble calfskin collagen, gelatin, and poly(proline) II in solution have been extended into the vacuum ultraviolet region. The extended spectrum of gelatin reveals that the circular dichroism of this unordered polymer is more closely related to the spectrum of charged polypeptides than might be evident from near ultraviolet work. A short-wavelength band is found at about 172 nm, which corresponds in position, magnitude, and sign to a band recorded earlier for poly(L -glutamic acid) at pH 8.0. This band is observed in a helical structure for the first time in the vacuum ultraviolet circular dichroism and absorption spectra of poly(proline) II. Both circular dichroism and absorption spectra point to the assignement of this band as the nσ*. Neither the nσ* nor the expected positive lobe of the ππ* helix band is observed in the extended circular dichroism spectrum of collagen. We postulate that these two bands cancel here in analogy to the case of α-helical poly(L -glutamic acid).     

Stepwise modification of the electrostatic charge of cytochrome c. Effects on protein conformation and oxidation-reduction properties

Horse heart cytochrome c was progressively maleylated, and fractions containing increasing numbers of modified lysines were obtained. The 695 nm band was present in derivatives containing up to 14 maleylated residues. Circular dichroic spectra showed minor changes beginning with 8 substituted lysines; in derivatives with 14 or more maleylated lysines, circular dichroism indicated total disruption of the native conformation. The ionic strength dependence of the measured oxidation reduction potentials and second order rate constants of reduction with ascorbate varied as expected from application of Debye-Huckel theory to the differently charged derivatives. The thermodynamic oxidation-reduction potentials decreased with the increase in the number of negatively charged groups, in a manner similar to that observed for simple iron complexes.     

Circular dichroism of holo- and apoprotocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

Circular dichroism studies have been carried out on both apo- and holoprotocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa, in the absence and presence of competitive inhibitors, protocatechualdehyde and 4-nitrocatechol. The apo- and holoenzyme showed identical spectra in the ultraviolet region between 200 and 250 nm (peptide back bone region), but the low intensity negative bands at 330 and 480 nm of the holoenzyme were completely absent in the apoenzyme. On the side chain region, the positive ellipticity peaks of the holoenzyme change into a lower intensity and broader band indicating the participation of aromatic amino acid residues in the primary binding of iron ion. Under anaerobic conditions, spectral changes were evident in the side chain region for the binary complexes of both the holo- and the apoenzyme with protocatechuate. The presence of iron in the holoenzyme results in an increase in positive ellipticity between 290 and 320 nm. Either with or without the iron, the enzyme protein binds protocatechuate and has a greater positive circular dichroism increase at 240-260 nm. CD difference spectra indicate that the modes of binding to form the binary complexes of holo- or apoenzyme with either substrates or competitive inhibitors are different. The bound iron ion stimulates binding. Spectral changes of the holoenzyme in the aromatic region were also observed in different pH environments of lower enzymatic activity. It is still not established whether these aromatic residues play an active or passive role in the binding of iron and/or substrates and inhibitors.     

Synthesis,resolution, and chiroptical properties of hemicryptophane cage controlling the chirality of propeller arrangement of a C3 triamide unit

The five‐steps synthesis of a hemicryptophane cage combining a benzene‐1,3,5‐tricarboxamide unit and a cyclotriveratrylene (CTV) moiety is described. Chiral high‐performance liquid chromatography (HPLC) was used to resolve the racemic mixture. The absolute configuration of the isolated enantiomers was assigned by comparison of the experimental electronic circular dichroism (ECD) spectra with the calculated ones. X‐ray molecular structures reveal that the capped benzene‐1,3,5‐tricarboxamide unit adopts a structurally chiral conformation in solid state: the chirality of CTV moiety controls the Λ or Δ orientation of the three amides.     

Circular dichroism studies on human chorionic somatomammotropin

The conformation of human chorionic somatomammotropin has been studied by means of circular dichroism spectra. The protein appears to contain about 45% α-helix in the native state. Circular dichroism bands in the region of side chain absorption have been assigned to phenylalanine and tryptophan residues. Tentative assignments has also been made to bands probably arising mostly from tyrosine residues. The stability of the native structure has been assessed by challenging the protein with four perturbing solvents. With the exception of 0.1 n NaOH which produced permanent denaturation, all conformational changes produced by the perturbants were fully reversible. In addition, the monomer molecular weight has been evaluated by gel filtration and osmotic pressure measurements. A value of 21,600 ± 900 was found by osmotic pressure at pH 8.4. The results have been compared with similar findings on human pituitary growth hormone and ovine pituitary lactogenic hormone.     

Circular dichroism of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus

Circular dichroism (CD) spectra of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus exhibit three positive ellipticity bands between 240 and 300 nm (250, 283, and 292 nm), two negative bands at 327 and 480 nm, and a low-intensity positive band at 390 nm. The fractions of helix β-form, and unordered form of the enzyme are 8, 38, and 54%, respectively. The circular dichroic bands at 327 and 480 nm and a part of the positive bands at 292 and 390 nm are associated with enzyme activity. Significant changes in absorption and CD spectra of the enzyme were observed when the temperature of the enzyme preparation was increased to 47°C, coinciding with the sharp decrease in enzyme activity observed at this temperature.     

Furoic acid derivatives from the endophytic fungus Coniothyrium sp.

The endophytic fungus Coniothyrium sp. was isolated from leaves of Quercus robur. Fermentation of this fungus on solid rice medium yielded two new furoic acid derivatives ( 1 and 2 ) and two additional known compounds. The structures of the new compounds were determined by extensive analysis of 1D and 2D nuclear magnetic resonance spectra as well as high-resolution mass spectrometry data. Compound 1 , containing three aromatic chromophores attached by rotatable sigma bonds and a chirality center in benzylic position, was found to be a scalemic mixture with an excess of the (S) enantiomer, the absolute configuration of which was elucidated as by the solution time-dependent density functional theory-electronic circular dichroism approach. The ωB97X/TZVP PCM/MeCN and SOGGA11-X/TZVP SMD/MeCN methods were used for geometry reoptimization to reproduce the solution conformational ensemble. All isolated compounds were tested for their cytotoxicity but proved to be inactive.     

Molecular biology of copper. A circular dichroism study on copper complexes of thionein and penicillamine.

Chicken liver Cd, Zn-thionein (metallothionein) was isolated from Cd-pretreated chickens weighing 1 500 g. The native Cd, Zn-thionein contained 9 g-atoms of metals per 12 000 g of protein. Upon the addition of Cu(CH3CN)4ClO4, all Cd2 and Zn2 were successfully replaced. 15 g-atoms of Cu from the acetonitrile perchlorate complex were bound to the protein. Due to the absence of aromatic amino acid residues, thionein has unique ultraviolet and circular dichroism properties. The shoulder of the ultraviolet spectrum at 250 nm (A250 X A280(-1) = 23.9) was shifted to 275 nm (A250 X A280(-1) = 1.6). No significant absorption was detected in the visible region. Th conformational changes of the protein moiety were much more visible in the circular dichroism spectra. The titration with Cu(CH3CH)2 caused the appearence of three new Cotton effects: 257.5 nm (+), 350 nm (+) and 301 nm (-). The negative Cotton effect at 239 nm of the original metallothionein was completely levelled off. The binding strength of copper with thionein is extraordinarily high: it survives proton treatment up to pH 1.9. Displacement of the Cd2 by Cu employing Cd-thionein which was formed at pH 2.2 resulted in the same circular dichroism properties as observed for Cu-thionein. D-Penicillamine proved a suitable model for the metal-free thionein, since redox reactions and polymerization of the sterically hindered thiol residue are known to be slow. The correlation of the circular dichroism properties of either copper complex using thionein or D-penicillamine was surprisingly high. Circular dichroism measurements of Cu(I)-D-penicillamine revealed Cotton effects at 255 nm (+), 280 nm (+) and 355 nm (-). Upon examining the red-violet mixed Cu(-i)-cu(II)-D-penicillamine complex, Cotton bands in the visible region at 425 nm (-) and 495 nm (+) were seen. In many blue copper enzymes, the copper is assumed to be in the neighborhood of both cysteine and aromatic amino acid residues, which are known to play an important role in the electron transfer. This is not the case in the Cu-thionein, which would explain many different properties of this copper protein. It is very attractive to conclude that the sterically hindered SH-group of D-penicillamine reacts with excess copper in a specific way, similar to the Cu-thionein. This phenomenon could explain the considerable success of D-penicillamine in the treatment of Wilson's disease.