Diamide model for the optical activity of collagen and polyproline I and II

A diamide, N-acetyl-L -proline-N,N-dimethylamide (AcProDMA), in water solution has optical rotatory dispersion (ORD) and circular dichroism (CD) spectra very similar to those of poly-L -proline II and the fibrous protein collagen. In contrast, AcProDMA in cyclohexane solution has optical activity resembling that of poly-L -proline I. Conformational analysis shows that AcProDMA is confined by steric constraints to either of two narrow regions of conformational space. The trans isomer of AcProDMA assumes conformations near those of polyproline II and collagen nearest neighbors, while cis-AcProDMA assumes conformations near that of polyproline I nearest neighbors. Nuclear magnetic resonance (NMR) experiments show that an equilibrium mixture of the cis and trans isomers of AcProDMA is present in solution. The trans isomer predominates in aqueous solution, but the equilibrium shifts to favor the cis isomer in nonpolar organic solvents such as cyclohexane. Analysis of the ORD spectra in terms of two basic spectra reveals a solvent dependent isomerization which parallels that observed by NMR. The optical activity of the pure isomers of AcProDMA can be derived from the ORD, CD and NMR data. A comparison of component cotton effects confirms the similarity in optical activity of trans-AcProDMA, polyproline II, and collagen on the one hand, and of cis-AcProDMA and Polyproline I on the other.     

The molecular structure of a curl-shaped retinal isomer

Computational studies of retinal protonated Schiff base (PSB) isomers show that a twisted curl-shaped conformation of the retinyl chain is a new low-lying minimum on the ground-state potential energy surface. The curl-shaped isomer has a twisted structure in the vicinity of the C₁₁=C₁₂ double bond where the 11-cis retinal PSB isomerizes in the rhodopsin photoreaction. The twisted configuration is a trapped structure between the 11-cis and all-trans isomers. Rotation around the C₁₀–C₁₁ single bond towards the 11-cis structure is prevented by steric interactions of the two methyl groups on the retinyl chain and by the torsion barrier of the C₁₀–C₁₁ bond in the other direction. Calculations of spectroscopic properties of the 11-cis, all-trans, and curl-shaped isomers provide useful data for future identification of the new retinal PSB isomer. Circular dichroism (CD) spectroscopy might be used to distinguish between the retinal PSB isomers. The potential energy surface for the orientation of the β-ionone ring of the 11-cis retinal PSB reveals three minima depending on the torsion angle of the β-ionone ring. Two of the minima correspond to 6-s-cis configurations and one has the β-ionone ring in 6-s-trans position. The calculated CD spectra for the two 6-s-cis configurations differ significantly indicating that the sign of the β-ionone ring torsion angle could be determined using CD spectroscopy. Calculations of the CD spectra suggest that a flip of the β-ionone ring might occur during the first 1 ps of the photoreaction. Rhodopsin has a negative torsion angle for the β-ionone ring, whereas the change in the sign of the first peak in the experimental CD spectrum for bathorhodopsin could suggest that it has a positive torsion angle for the β-ionone ring. Calculated nuclear magnetic resonance (NMR) shielding constants and infrared (IR) spectra are also reported for the retinal PSB isomers. Figure The figure shows the optimized molecular structure of the curl-shaped retinal isomer. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Association equilibrium of d-methamphetamine and l-methamphetamine with serum albumin

The association equilibria for complex formation between serum albumin (bovine, rat) and the optical isomers of methamphetamine (MAMP) was determined using an ultrafiltration method. It was found that serum albumin/d-MAMP and serum albumin/l-MAMP complexes had distinctly different Scatchard plots with bovine and rat albumin. The binding parameters of each association equilibrium were estimated from the Scatchard plots by Rosenthal's graphic method. This distinguished two kinds of specific binding sites in terms of the association equilibrium between bovine serum albumin and d-MAMP, and one binding site for rat serum albumin and d-MAMP. One specific binding site was found between serum albumin and l-MAMP in both bovine and rat. Molar ellipticities, [θ], of peaks were decreased in the CD spectra of the complexes formed between bovine serum albumin and d-MAMP or l-MAMP when compared with the CD spectrum of bovine serum albumin alone. However, no difference in [θ] was found between the CD spectra of the enantiomers of MAMP in the measured wavelength range. The non-specific binding site was distinct from the specific binding site and resulting from altered tertiary structure of the albumin molecule. Chirality 10:742–746, 1998. © 1998 Wiley-Liss, Inc.     

Differentiation of isomeric malonylated flavonoid glyconjugates in plant extracts with UPLC-ESI/MS/MS

Introduction: Glycosylation at different hydroxyl groups of flavonoids and acylation of sugar moieties are ubiquitous modifications observed in plants. These modifications give rise to simultaneous presence of numerous isomeric and isobaric compounds in tissues and extracts thereof. Objective: To develop UPLC‐MS method capable for resolution of isomeric malonylated glycoconjugates of flavonoids and recognition of structural differences. Methodology: Flavonoid glycoconjugates were extracted from leaves of blue lupin (Lupinus angustifolius L.) plants with 80% methanol. Extracts were analysed using ultraperformance liquid chromatography (UPLC) combined with tandem (quadrupole–time of flight, QToF) mass spectrometry. Results: Differentiation of malonylated glycosides of isoflavones and flavones is demonstrated in this paper. The use of UPLC‐MS/MS enabled 38 flavonoid conjugates to be distinguished, including the discrimination of five different isomers of a single 3′‐O‐methylluteolin glycoside. Additionally, pseudo MS³ experiments (CID spectra registered at high cone voltages) enabled confirmation of the aglycone structures by comparison of their spectra with those obtained from aglycone standards. Conclusions: Application of UPLC‐MS/MS allows separation and identification numerous positional isomers of malonylated glycosides of flavonoids and isoflavonoids in plant material. Provided there is strict control of the MS ionisation parameters, this method may be useful for preparation of a flavonoids spectra database, enabling the inter‐laboratory comparison of analytical results. Copyright © 2008 John Wiley & Sons, Ltd.     

Sequence dependence of the circular dichroism of synthetic double-stranded RNAs

We have synthesized and studied the CD spectra of five new double-stranded RNA polymers: poly[r(A-G)·r(C-U)], poly[r(A-U-C)·r(G-A-U)], poly[r(A-C-U)·r(A-G-U)], poly[r(A-A-C)·r(G-U-U)], and poly[r(A-C-C)·r(G-G-U)]. Together with previously published spectra of seven other RNA sequences, the spectra of these new sequences provide a library sufficient to approximate the spectra of all other RNA sequences by first-neighbor formulas and, in addition, give four spectra with which we may test the validity of first-neighbor approximations. (1) We find that the spectra of RNA sequence isomers are very different, but that the spectra essentially do obey first-neighbor relationships. (2) We have derived tentative first-neighbor assignments of negative bands at about 295 and 210 nm in the CD spectra. (3) A test of spectral independence shows that among the 12 polymer spectra there are at least seven significant independent spectral shapes, one less than the eight needed to give the most accurate spectral analysis of an unknown RNA sequence for its first-neighbor frequencies. (4) Spectra are calculated for RNAs of random base composition, approximating natural RNAs having complex sequences. (5) A T-matrix of spectral components assigned to the first-neighbor base pairs is derived from 10 of the spectra. This matrix allows an estimation of the CD spectrum of any other known RNA sequence or an analysis of the spectrum of an unknown sequence for its distribution of first-neighbor base-pair frequencies. (6) Test analyses of two of the synthetic polymers and of two natural RNAs set a probable limit on the accuracy of first-neighbor frequency determinations using this T-matrix. (7) Finally, we summarize in an appendix the melting temperatures for all the RNA and corresponding DNA sequences; it appears that the T_m values of both DNAs and RNAs approximately obey first-neighbor relationships.     

Triterpenoids from the Stem Bark of Melia toosendan and Determination of Their Absolute Configurations at C(24)

Six new triterpenoids, meliasenins S–X ( 1 – 6 , resp.), were isolated from the stem bark of Melia toosendan. Their structures were elucidated by mass spectrometry, NMR experiments, and comparison with the known compounds. Particularly, the absolute configuration at C(24) in new compounds was determined through their CD spectra of the [Pr(FOD)₃] complex (fod=1,1,1,2,2,3,3,7,7,7‐decafluoroheptane‐4,6‐dione) in CCl₄, as well as by using Mosher's method.     

Resolution of optical isomers by high-pressure liquid chromatography: The separation of benzo[a]pyrene trans-diol derivatives

The optical isomers of (±)r-7,t-8-dihydroxy-7,8-dihydrobenzo[a]pyrene and its synthetic precursor (±)r-7,t-8-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene were resolved as their di-(−)menthoxyacetates using high-pressure liquid chromatography. Saponification of the resolved diesters yielded the corresponding enantiomers. The specific rotation, CD spectra, and ORD curves are reported. The resolution of these optical isomers permits detailed studies on the enzymatic intermediates and the mechanism of benzo[a]pyrene activation to its carcinogenic form. The method is of general usefulness for the resolution of optical isomers.     

Circular dichroism of green bacterial chlorosomes

Positive and negative bands in previously measured circular dichroism (CD) spectra of Chlorobium limicola chlorosomes appeared to be sign-reversed relative to those of Chloroflexus aurantiacus chlorosomes in the 740–750 nm spectral region where bacteriochlorophyll (BChl) c absorbs maximally. It was not clear, however, whether this difference was intrinsic to the chlorosomes or was due to differences in the procedures used to prepare them. We therefore repeated the CD measurements using chlorosomes isolated from both Cb. limicola f. thiosulfatophilum and Cf. aurantiacus using the method of Gerola and Olson (1986, Biochim. Biophys. Acta 848: 69–76). Contrary to the earlier results, both types of chlorosomes had very similar CD spectra, suggesting that both have similar arrangements of BChl c molecules. The previously reported difference between the CD spectra of Chlorobium and Chloroflexus chlorosomes is due to the instability of Chlorobium chlorosomes, which can undergo a hypsochromic shift in their near infrared absorption maximum accompanied by an apparent inversion in their near infrared CD spectrum during isolation. Treating isolated chlorosomes with the strong ionic detergent sodium dodecylsulfate, which removes BChl a, does not alter the arrangement of BChl c molecules in either Chloroflexus or Chlorobium chlorosomes, as indicated by the lack of an effect on their CD spectra.Abbreviations BChl bacteriochlorophyll - Cb. Chlorobium - CD circular dichroism - Cf. Chloroflexus - NIR near infrared     

Identification of methylated flavonoid regioisomeric metabolites using enzymatic semisynthesis and liquid chromatography-tandem mass spectrometry

Discrimination of isomeric methylated metabolites is an important step toward identifying genes responsible for methylation, but presents substantial challenges because authentic standards are often unavailable and mass spectra of isomers have been considered indistinguishable. In this report, an approach is described for identifying methyl group positions in multiply methylated flavonoid metabolites using combinations of tandem mass spectrometry, liquid chromatography retention, and site-selective methylation by recombinant O-methyltransferases from Solanum habrochaites LA1777. The basis for observed fragment ions in tandem mass spectra of multiply methylated myricetin was further established using enzymatic incorporation of deuterium-labeled methyl groups using S-adenosylmethionine-d ₃ as precursor.

     

Random-phase calculation of the low-energy circular dichroism in glucans

The CD of the long-wavelength electronic transition in the α-(1 → 4)-linked glucan dimer (maltose) and polymer (amylose), and in the β-(1 → 6)-linked dimer (gentiobiose) and polymer (pustulan), are calculated in the random-phase approximation using time-dependent Hartree theory. This long-wavelength transition (180–190 nm) is assumed to be localized on the linkage oxygen atom and to be of a σ*/3 s ← n character. The zerothorder σ*/3s ← n magnetic-dipole transition moment is coupled to the CC, CO, and CH bond density of states via a polarizability approximation. We assume this coupling is dominated energetically by the electric-dipole, electric-dipole interaction terms in the context of Schellman's μ_e-μ_m coupling mechanism of rotatory power. The CD is calculated as a function of rotation about the two single bonds of the linkage oxygen atom and also as a function of rotation about the C(5)-C(6) bond. For maltose, four rotational isomers are considered, resulting from combinations of the gg and gt C(6)H₂OH group rotational isomers. The calculated CD values were then Boltzmann-averaged over an empirical potential, and the resulting CD was found to compare satisfactorily with experiment. In the case of the polymers, only structures having periodicity (helicity) were examined.     

A specific stain for α-glucosidases in isoelectric focusing gels

The separation of the four diastereomers of β,γ-bidentate Cr · ATP using reverse-phase HPLC techniques is described. This technique provides complete resolution of the diastereomers within 10 min and relies on the use of methanesulfonic acid (in the ionized form) as an ion-pairing agent. To identify the screw sense of these resolved isomers, the CD spectra of each isomer were done, and substrate and inhibition specificities were examined using hexokinase. The results were then correlated with the isomeric assignments made by D. Dunaway-Mariano and W. W. Cleland (1980, Biochemistry19, 1506–1515). Further studies included the monitoring of isomer interconversion at pH 6.2 to an equilibrium concentration of all four, and specific rotation measurements of the pure isomers at pH 2.5, 23°C, and 546 nm.     

Conformation and stability of streptokinases from nephritogenic and nonnephritogenic strains of streptococci

Conformation and stability of three Sks from Streptococcus equisimilis strain H46A, Streptococcus pyogenes strain A374, and Streptococcus pyogenes strain AT27 were compared by limited proteolysis, CD, and fluorescence measurements and by DSC. The general similarity of the peptide CD spectra in the spectral region 185 to 260 nm indicates the same type of folding for the three proteins. Fluorescence and aromatic CD spectra are consistent with a predominant surface localization of the aromatic amino acids and a low rigidity of their surroundings. A major difference among the three Sks is shown by deconvolution of their excessive heat capacity functions. Deconvolution reveals two energetic folding units in Sk H46A but three energetic folding units in Sk A374 and Sk AT27. Digestion of the Sks with trypsin indicates a reduced sensitivity of the C-terminal region of Sk A374 and Sk AT27 in comparison to Sk H46A. This suggests that amino acids of the C-terminal region participate in the formation of the third folding unit of Sk A374 and Sk AT27. Proteins 27:26–35 © 1997 Wiley-Liss, Inc.     

Circular dichroism of N-phenylnaphthylamine derivatives complexed with the β-form of poly(L-lysine)

Complex formation of 1-anilinonaphthalene-8-sulfonate (ANS) and 2-p-toluidinonaphthalene-6-sulfonate (TNS) with the β-form of poly(L -lysine) [(β-Lys)_n] has been studied by circular dichroism (CD) and absorption spectra measurements. Not only hydrophobic interactions but also hydrogen-bonding and electrostatic interactions contribute to complex formation. The relative importance of these stabilizing factors depends on the relative position of the arylamino group and the sulfonate. For example, ionic interactions play a significant role in the binding of 1,8-ANS and 1,8-TNS, but not in the case of 2,6-TNS. The induced CD of the complexes of (β-Lys)_n with 1,8-ANS and 1,8-TNS is consistent with theoretical calculations for nonplanar conformations of these dyes, twisted in a left-handed sense. As expected for steric reasons, the dominant isomer is one in which the arylamino group is oriented away from the 8-sulfonate (α₁). The induced CD of complexes with 2,6-TNS can be accounted for by an equimolar mixture of left-handed isomers in which the arylamino group is oriented toward the 1-position (β₂) and toward the 3-position (β₁). Our results demonstrate that (β-Lys)_n is capable of chiral discrimination and suggest its general utility for CD studies of racemic anionic dyes.     

Stability,toxicity and biological activity of retro,inversed and retro‐inversed glucagon isomers

Peptide modifications that improve pharmacological properties are of considerable therapeutic importance. Here we consider the retro (R), inversed (D) and retro‐inversed (RI) isomers of glucagon with respect to structure, stability, toxicity and biological activity. Biologically, RI‐glucagon demonstrated comparable in vivo activity as L‐glucagon with respect to magnitude and duration of blood sugar elevation following i.p. administration to mice. Structurally, the isomers were investigated through circular dichroism (CD) and nanopore analysis. CD demonstrated a conserved potential for formation of secondary structure, which was independent of the direction of the peptide (L vs R; D vs RI) as well as formation of symmetry‐related structures for the chiral isomers (L vs D; R vs RI). CD, therefore, discriminated chiral but not directional isomers. Nanopore analysis, which depends on interaction of the peptides with chiral pores, discriminated all four isomers on the basis of unique signatures of bumping and translocation. Nanopore analysis offered greater opportunity than CD to discriminate the isomers although neither technique provided a definitive biomarker of biological activity. Functionally, the R and RI isomers resist proteolytic degradation and none of the isomers possess hemolytic activity or cellular toxicity. Collectively, this investigation highlights the potentials and limitations of CD and nanopore analysis for investigation of peptide isomers as well as offering insight into the structural criteria to mimic peptide biological activity. For this example, retro‐inversion, through undefined contributions of increased stability and maintained biological activity, was best suited to mimic the biological activity of the parent peptide. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Basic polypeptides as histone models. Synthesis, conformation, and interaction with DNA of statistical copolymers (Lys x,Ala y)n.

Statistical copolymers (Lys^x,Ala^y)_n were synthesized by copolymerization of N-carboxyanhydrides of L -amino acids. The conformation of copolymers in aqueous solutions was investigated using circular dichroism (CD). Calculations based on the CD data showed that polymers (Lys^x,Ala^y)_n can exhibit a random conformation, an α-helix, and a β-structure in various ratios. CD spectra of complexes of copolymers with DNA prepared by gradual dialysis from a high ionic strength to 0.15 M NaCl can be correlated with the copolymer conformation in medium and high ionic strength. For copolymers forming an α-helix and β-structure, these spectra show resemblance with similar spectra of complexes of those histones that are able to exhibit ordered conformations.     

Structural insights into the inclusion complexes between clomiphene citrate and β‐cyclodextrin: The mechanism of preferential isomeric selection

A major challenge in pharmaceuticals for clinical applications is to alter the solubility, stability, and toxicity of drug molecules in living systems. Cyclodextrins (CDs) have the ability to form host–guest inclusion complexes with pharmaceuticals for further development of new drug formulations. The inclusion complex of clomiphene citrate (CL), a poorly water‐soluble drug, with native β‐cyclodextrin (β‐CD) was characterized by a one and two‐dimensional nuclear magnetic resonance (NMR) spectroscopic approach and also by molecular docking techniques. Here we report NMR and a computational approach in preferential isomeric selection of CL, which exists in two stereochemical isomers, enclomiphene citrate (ENC; E isomer) and zuclomiphene citrate (ZNC; Z isomer) with β‐CD. β‐CD cavity protons, namely, H‐3′ and H‐5′, experienced shielding in the presence of CL. The aromatic ring protons of the CL molecule were observed to be deshielded in the presence of β‐CD. The stoichiometric ratio of the β‐CD:CL inclusion complex was observed by NMR and found to be 1:1. The overall binding constant of β‐CD:CL inclusion complexes was based on NMR chemical shifts and was calculated to be 50.21 M⁻¹. The change in Gibb's free energy (∆G) was calculated to be −9.80 KJ mol⁻¹. The orientation and structure of the β‐CD:CL inclusion complexes are proposed on the basis of NMR and molecular docking studies. 2D ¹H‐¹H ROESY confirmed the involvement of all three aromatic rings of CL in the inclusion complexation with β‐CD in the solution, confirming the multiple equilibria between β‐CD and CL. Molecular docking and 2D ¹H‐¹H ROESY provide insight into the inclusion complexation of two isomers of CL into the β‐CD cavity. A molecular docking technique further provided the different binding affinities of the E and Z isomers of CL with β‐CD and confirmed the preference of the Z isomer binding for β‐CD:CL inclusion complexes. The study indicates that the formation of a hydrogen bond between –O– of CL and the hydrogen atom of the hydroxyl group of β‐CD was the main factor for noncovalent β‐CD:CL inclusion complex formation and stabilization in the aqueous phase.     

Application of (S)‐TBMB carboxylic acid‐based on‐line HPLC‐exciton CD analysis to acyclic vicinal alcohols and amino alcohols

Applications of the on‐line HPLC‐exciton CD analysis using (S)‐2‐tert‐butyl‐2‐methyl‐1,3‐benzodioxole‐4‐carboxylic acid [(S)‐TBMBC‐OH] that can simultaneously determine the enantiomeric compositions and the absolute configuration of cyclohexane‐1,2‐diols and diamines as well as acyclic vicinal diols and amino alcohols were studied. Di‐O‐ or di‐N,O‐(S)‐TBMBC derivatives of acyclic terminal vicinal diols, 2‐hydroxy‐1‐amines, and nonterminal vicinal diols gave symmetrical exciton CD spectra between enantiomers, indicating their absolute configurations. However, Di‐N,O‐(S)‐TBMBC derivatives of 2‐amino‐1‐ols did not always give symmetrical exciton CD spectra between enantiomers, but their 2‐phthalimido‐1‐O‐(S)‐TBMBC derivatives gave symmetrical exciton CD spectra, indicating their absolute configurations. All these (S)‐TBMBC derivatives were separated by normal‐phase HPLC and unequivocally determined by the on‐line HPLC‐exciton CD analysis without recourse to reference samples. Chirality 11:149–159, 1999. © 1999 Wiley‐Liss, Inc.     

Conformational differences between cis and trans proline isomers of a peptide antigen representing the receptor binding domain of Pseudomonas aeruginosa as studied by 1H-nmr

A 17-residue disulfide-bridged peptide (PAK 128–144) corresponding to the C-terminus of Pseudomonas aeruginosa pilin strain K has been studied by one- and two-dimensional nmr techniques. This synthetic immunogen has been found to exist as two distinct conformations in solution, which have been demonstrated to arise as a result of the isomerization of the I¹³⁸-P¹³⁹ amide bond. The two isomers occur in the ratio of 3 : 1 trans to cis at 5°C. Sequential assignments for both forms have been accomplished through the use of nuclear Overhauser enhancement spectroscopy (NOESY) spectra and most side-chain resonances have been assigned using a combination of correlated spectroscopy, total correlated spectroscopy, and NOESY spectra. The presence of the cis isomer, which is considerably more predominant in the oxidized peptide, was confirmed by the observation of the characteristic NOEs between P¹³⁹ and the preceding residue. Further corroboration was given by the disappearance of the cis resonances in the spectrum of the P139A analogue of PAK 128–144. From observation of the differences in the chemical shifts and amide proton temperature coefficients of the two isomers, it is apparent that the two forms differ markedly in their solution conformation. The biological implications of the isomerization are discussed. © 1994 John Wiley & Sons, Inc.     

La Préparation et l’isomérisme géometrique des Acides Cinnamiques Méthylthio-α Substitués

An improved method of preparation and the separation of geometrical isomers of cis and trans α-methylthio-cinnamic acid derivatives are described. The observation on the NMR spectra and the determination of geometrical structure of these compounds by various spectrometric methods are also included.     

Interaction of poly-5-bromouridylic acid. I. Formation of helical complexes with various adenine and 2-aminoadenine derivatives

Complexes of poly(BU) with various adenine derivatives were investigated by circular dichroism (CD) and absorption spectroscopy. A 1:2 stoichiometry was indicated on CD mixing curves for typical complexes of 9-substituted adenine and 2-aminoadenine derivatives with poly(BU). The CD spectrum of adenosine·2poly(BU) is characterized by well-resolved bands in the range of 210–350 nm. Other adenine derivative–poly(BU) complexes also afford similar CD spectra, while 2-aminoadenine derivative–poly(BU) complexes give quite different spectra. Attempts to assign representative CD spectra were made using the transition of helical poly(BU) and the respective purine polynucleotides. The similarity of the CD spectra suggests that poly(A)·2poly(BU) and adenine derivative–poly(BU) complexes are nearly identical in structure except for the ribose–phosphate linkage. The fact that the uv isosbestic point of adenosine·2poly(BU) falls in close proximity to that of the corresponding polymer complex also supports this conclusion. In the formation of stable helices, the ribose moiety is dispensable in the “strand” of purine. The T_m of 9-methyladenine·2poly(BU) is somewhat higher than that of adenosine·2poly(BU) under equivalent conditions. The T_m difference with the monomer–poly(U) system was found to be about 20°C in 0.4M NaCl–0.02M Na–cacodylate–5 × 10^?4M EDTA (pH 7.0). Further, it was noted that the monomer–poly(BU) complexes are formed even when the T_m is lower than that of self-folded poly(BU).