Characteristic Circular Dichroism Spectra Research Articles

Advantages of Induced Circular Dichroism Spectroscopy for Qualitative and Quantitative Analysis of Solution-Phase Cyclodextrin Host-Guest Complexes.

The presence of a chiral or chirally perturbed chromophore in the molecule under investigation is a fundamental requirement for the appearance of a circular dichroism (CD) spectrum. For native and for most of the substituted cyclodextrins, this condition is not applicable, because although chiral, cyclodextrins lack a chromophore group and therefore have no characteristic CD spectra over 220 nm. The reason this method can be used is that if the guest molecule has a chromophore group and this is in the right proximity to the cyclodextrin, it becomes chirally perturbed. As a result, the complex will now provide a CD signal, and this phenomenon is called induced circular dichroism (ICD). The appearance of the ICD spectrum is clear evidence of the formation of the complex, and the spectral sign and intensity is a good predictor of the structure of the complex. By varying the concentration of cyclodextrin, the ICD signal changes, resulting in a saturation curve, and from these data, the stability constant can be calculated for a 1:1 complex. This article compares ICD and NMR spectroscopic and molecular modeling results of cyclodextrin complexes of four model compounds: nimesulide, fenbufen, fenoprofen, and bifonazole. The results obtained by the different methods show good agreement, and the structures estimated from the ICD spectra are supported by NMR data and molecular modeling.

Proteomic and Transcriptome Profiling of G-Quadruplex Aptamers Developed for Cell Internalization.

Nucleic acid medicine is expected to be among the most promising next-generation therapies. Applications of nucleic acid in vivo are still challenging as a result of the difficulties in direct cell penetration without external assistance. To facilitate the cellular delivery of therapeutic nucleic acid, we developed cell-penetrating aptamers using cell-internalization Systematic Evolution of Ligands by EXponential enrichment (SELEX). Moreover, C20-4 min, a G-quadruplex-forming DNA aptamer, was discovered, showing a higher cell-penetrating capacity compared with other candidates, including AS1411. To verify the formation and understand the G-quadruplex folding topologies of enriched aptamer motifs, characteristic circular dichroism (CD) spectral features are analyzed. The CD spectra of C20-4 min strongly support the formation of parallel G-quadruplexes. Systematic analyses of the G-quadruplex regulation pathway have been performed by combining aptamer pull-down with mass spectrometry. We profiled G-quadruplex aptamers interacting with cellular proteins during internalization and identified helicases and GTPase proteins as cellular interacting partners. In addition, whole transcriptome analysis was performed to study the effects of G-quadruplex aptamers, revealing differentially expressed genes involved in the regulation of GTPase functions. Integrative analyses of transcriptome and proteomic have aided in understanding the functional hierarchy of molecular players in G-quadruplex nucleic acid mechanisms of internalization, which might facilitate developing a novel delivery system.

Adaptive Chirality of an Achiral Cucurbit[8]uril‐Based Supramolecular Organic Framework for Chirality Induction in Water

AbstractChiral framework materials have been developed for many applications including chiral recognition, chiral separation, asymmetric catalysis, and chiroptical materials. Herein, we report that an achiral cucurbit[8]uril‐based supramolecular organic framework (SOF‐1) with the dynamic rotational conformation of tetraphenylethene units can exhibit adaptive chirality to produce M‐SOF‐1 or P‐SOF‐1 with mirror‐image circular dichroism (CD) with gabs≈±10−4 and circularly polarized luminescence (CPL) with glum≈±10−4 induced by L‐/D‐phenylalanine in water, respectively. The chirality induction in CD (gabs≈−10−4) and CPL (glum≈−10−4) of P‐SOF‐1 from achiral SOF‐1 can be presented by using a small amount of adenosine‐5′‐triphosphate disodium (ATP) or adenosine‐5′‐diphosphate disodium (ADP) (only 0.4 equiv) in water. Furthermore, the adaptive chirality of SOF‐1 can be used to determine dipeptide sequences (e.g., Phe‐Ala and Ala‐Phe) and distinguish polypeptides/proteins (e.g., somatostatin and human insulin) with characteristic CD spectra. Therefore, achiral SOF‐1 as an ideal chiroptical platform with adaptive chirality may be applied to determine the enantiopurity of amino acids (e.g., L‐/D‐phenylalanine), develop aqueous CPL materials, and distinguish biological chiral macromolecules (e.g., peptides/proteins) via chirality induction in water.

Adaptive Chirality of an Achiral Cucurbit[8]uril-Based Supramolecular Organic Framework for Chirality Induction in Water.

Chiral framework materials have been developed for many applications including chiral recognition, chiral separation, asymmetric catalysis, and chiroptical materials. Herein, we report that an achiral cucurbit[8]uril-based supramolecular organic framework (SOF-1) with the dynamic rotational conformation of tetraphenylethene units can exhibit adaptive chirality to produce M-SOF-1 or P-SOF-1 with mirror-image circular dichroism (CD) with gabs ≈±10-4 and circularly polarized luminescence (CPL) with glum ≈±10-4 induced by L-/D-phenylalanine in water, respectively. The chirality induction in CD (gabs ≈-10-4 ) and CPL (glum ≈-10-4 ) of P-SOF-1 from achiral SOF-1 can be presented by using a small amount of adenosine-5'-triphosphate disodium (ATP) or adenosine-5'-diphosphate disodium (ADP) (only 0.4 equiv) in water. Furthermore, the adaptive chirality of SOF-1 can be used to determine dipeptide sequences (e.g., Phe-Ala and Ala-Phe) and distinguish polypeptides/proteins (e.g., somatostatin and human insulin) with characteristic CD spectra. Therefore, achiral SOF-1 as an ideal chiroptical platform with adaptive chirality may be applied to determine the enantiopurity of amino acids (e.g., L-/D-phenylalanine), develop aqueous CPL materials, and distinguish biological chiral macromolecules (e.g., peptides/proteins) via chirality induction in water.

Chirality sensing of terpenes, steroids, amino acids, peptides and drugs with acyclic cucurbit[n]urils and molecular tweezers.

Achiral chromophoric hosts, i.e. acyclic cucurbit[n]urils and molecular tweezers, were found to respond with characteristic Circular Dichroism (CD) spectra to the presence of micromolar concentrations of chiral hydrocarbons, terpenes, steroids, amino acids and their derivates, and drugs in water. In favourable cases, this allows for analyte identification or for reaction monitoring.

Octarellin VI: Using Rosetta to Design a Putative Artificial (β/α)8 Protein

The computational protein design protocol Rosetta has been applied successfully to a wide variety of protein engineering problems. Here the aim was to test its ability to design de novo a protein adopting the TIM-barrel fold, whose formation requires about twice as many residues as in the largest proteins successfully designed de novo to date. The designed protein, Octarellin VI, contains 216 residues. Its amino acid composition is similar to that of natural TIM-barrel proteins. When produced and purified, it showed a far-UV circular dichroism spectrum characteristic of folded proteins, with α-helical and β-sheet secondary structure. Its stable tertiary structure was confirmed by both tryptophan fluorescence and circular dichroism in the near UV. It proved heat stable up to 70°C. Dynamic light scattering experiments revealed a unique population of particles averaging 4 nm in diameter, in good agreement with our model. Although these data suggest the successful creation of an artificial α/β protein of more than 200 amino acids, Octarellin VI shows an apparent noncooperative chemical unfolding and low solubility.

Theoretical and Experimental Studies of Circular Dichroism of Mono- and Diazonia[6]helicenes

Combined experimental and theoretical studies revealed the characteristic circular dichroism (CD) spectral profiles of mono- and diazonia[6]helicenes, which were distinctly different from those reported for parent [6]helicene and neutral (di)aza-analogues. Aza[6]helicenes and [6]helicene showed bisignate Cotton effects (CEs) at the (1)Ba and (1)Bb bands, along with a weak CE at the (1)Lb band, where the signs of the former bands are responsible for the helical chirality of the helicenes while the sign of the latter is susceptive to the various factors such as electronic and steric effects. Protonation to monoaza[6]helicenes produces azonia[6]helicenes, showing dramatic changes in the CE pattern from the two bisignate to a three positive, two negative CE extremum series of comparable magnitudes, while dual protonation to diaza[6]helicenes forming diazonia[6]helicenes led to only nominal changes (slightly different rotational strength and excitation energy) in the CE pattern. Such rather complicated and contrasting CE behaviors of mono- versus diazoniahelicenes are derived mostly from the electronic effects of (unsymmetrical) protonation because the structures of neutral, mono-, and dicationic species are essentially identical to each other. Compared with those of neutral (di)aza[6]helicenes, the experimental CD spectra of (di)azonia[6]helicenes were less satisfactorily reproduced by the theoretical calculations at the state-of-the-art RI-CC2/TZVPP//DFT-D2-B97-D/TZVP level, most probably due to the inadequate incorporation of the effects of solvation. Nevertheless, the bytheoretical predictions were reasonably accurate and highly valuable in assigning the observed CE and elucidating the origin of the elaborate CD spectral behaviors upon protonation through inspection of the molecular orbital configuration of each transition, encouraging the extended use of the present protocol for analyzing the CD spectral behavior of aza- and other heteroatom-incorporated helicenes upon protonation. The CD spectral behavior upon metal ligation will also be explained through further theoretical and experimental studies.

Characteristic oxygen K-edge circular dichroism spectra of amino acid films by improved measurement technique

Circular dichroism (CD) spectroscopy in the soft x-ray energy region is a new tool to study the local structure of chiral materials. In this paper, we introduce a method to measure high-quality CD spectra in the oxygen K-edge energy region. Characteristic CD spectra of thin films of the amino acids L-tyrosine and L-aspartic acid are reported and compared with those of films of L-alanine and L-serine. The signals from the oxygen 1s → π∗ transitions of COO-, which is a common moiety in these amino acids, reflect the local geometry of each amino acid.

A helically twisted imine macrocycle that allows for determining the absolute configuration of α-amino carboxylates

Binding of α-amino carboxylates to a helically twisted imine macrocycle based on the indolocarbazole scaffold gives rise to characteristic circular dichroism spectra, and the patterns of the Cotton effects are consistent with the absolute configuration of α-amino carboxylates.

Optical Activity of Achiral Ligand SCH3 Adsorbed on Achiral Ag55 Clusters: Relationship between Adsorption Site and Circular Dichroism

The electronic circular dichroism (CD) spectra of a methyl-thiol adsorbed at different sites on an icosahedral silver nanoparticle is studied by using time-perturbed density functional theory. Despite that separately molecule and nanoparticle are achiral and consequently optically inactive, the Ag(55)-SCH(3) compound emerges with a new symmetry, which may be chiral or not depending on the adsorption site and orientation of the molecule. It is found that chirality is favored when the thiol is adsorbed between two atoms of different coordination number. Chiral compounds have characteristic CD spectra in the UV-visible region, where Ag(55) shows optical absorption but SCH(3) does not; revealing that highly degenerated molecular-like electronic states of Ag(55) are modified by the presence of the molecule inducing optical activity. It is concluded that CD line-shapes and magnitude strongly depend on the site where the adsorption takes place, while its intensity is modulated by the molecule orientation.

Conformation Analysis of Soybean Protein in Reverse Micelles by Circular Dichroism Spectroscopy

Far-UV circular dichroism (CD) spectroscopy was used to study the conformation of protein from soybean flour under the influence of various reverse micelle environments and salt treatments, as compared with the protein using aqueous buffer extraction. These proteins exhibited characteristic CD spectra that reflected a considerable amount of residual secondary structures. The CD analysis showed that the protein modification by reverse micellar system was related to loss of α-helix and β-structure and increase of random coil. The ellipticity of protein in bis (2-ethylhexyl) sodium sulfosuccinate (AOT) and sodium dodecyl sulfate (SDS) reverse micelles was higher than in hexadecyl trimethyl ammonium bromide (CTAB) and TrionX-100. The CD spectra in reverse micelles except for SDS at 210 nm had lower intensity than in aqueous solution. Salts could influence the ellipticity of protein at near 194 nm in an order of NaCl > KNO3> > KCl > NaNO3 > Na2SO4 in monovalent salts and in MgCl2 > BaCl2 > CaCl2 divalent ones. Besides, the ellipticity was totally higher in monovalent salts than in divalent ones.

Circular dichroism of nucleoside derivatives. VII. The electronic structure and spectra of some simple nucleoside derivatives

The electronic structures and spectra of some simple nucleoside derivatives with the formal structure of cytosine nucleoside are presented. The experimental spectroscopic information is obtained from the circular dichroism and absorption spectra. The first four circular dichroism bands of pi-pi* origin possess the same sign pattern previously reported for most cytosine nucleoside derivatives. The four electronic states that produce the characteristic circular dichroism spectra are correlated with the B2u, B1u, and E1u states of benzene. Pariser-Parr-Pople calculations are also presented which qualitatively explain the trends in both the circular dichroism and absorption spectra. Configuration interaction involving single electron excitations between the five highest occupied and the three lowest unoccupied molecular orbitals has been included in the calculations.

Chiral Correlation between Low-Birefringent Phases with Twist Grain Boundary-like Helix and Highly Birefringent Phases with Layer Chirality as Elucidated from Circular Dichroism Observations

The bent-shaped molecule with a central naphthalene core, 2,7-naphthalene-bis[4-(4-dodecylphenyliminomethyl)]benzoate, forms the low-birefringent B2 (LB-B2) phase with the twist grain boundary (TGB)-like helical structure and the low-birefringent B4 (LB-B4) phase in order of decreasing temperature. By applying the electric field, the LB-B2 phase is altered to the highly birefringent B2 (HB-B2) phase because of unwinding of the TGB-like helix. The HB-B2 phase is transformed to the HB-B4 phase without a loss of birefringence on cooling. These four phases show characteristic circular dichroism spectra, showing the consistent correlation through the transformation between these phases. The source of the chirality in the achiral system and the correlation in the chirality between these phases are discussed.

Evidence of PPII-like Helical Conformation and Glass Transition in a Self-Assembled Solid-State Polypeptide−Surfactant Complex: Poly(l-histidine)/Docylbenzenesulfonic Acid

We present lamellar self-assembly of cationic poly(L-histidine) (PLH) stoichiometrically complexed with an anionic surfactant, dodecyl benzenesulfonic acid (DBSA), which allows a stabilized conformation reminiscent of polyproline type II (PPII) left-handed helices. Such a conformation has no intrapeptide hydrogen bonds, and it has previously been found to be one source of flexibility, e.g., in collagen and elastin, as well as an intermediate in silk processing. PLH(DBSA)1.0 complexes were characterized by Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC). The PPII-like conformation in PLH(DBSA)1.0 is revealed by characteristic CD and FTIR spectra, where the latter indicates absence of intrachain peptide hydrogen bonds. In addition, a glass transition was directly verified by DSC at ca. 135 degrees C for PLH(DBSA)1.0 and indirectly by SAXS and TEM in comparison to pure PLH at 165 degrees C, thus indicating plasticization. Glass transitions have not been observed before in polypeptide-surfactant complexes. The present results show that surfactant binding can be a simple scheme to provide steric crowding to stabilize PPII conformation to tune the polypeptide properties, plasticization and flexibility.

Experimental and Theoretical Study of the CD Spectra and Conformational Properties of Axially Chiral 2,2‘-, 3,3‘-, and 4,4‘-Biphenol Ethers

New chiral biphenol ethers (Biph22, Biph33, and Biph44), carrying (R)-methylpropyloxy groups at 2,2'-, 3,3'-, and 4,4'-positions of biphenyl, were prepared. The introduced peripheral chiral groups in these biphenol ethers induce an (averaged) axial chirality to give predominant aR- or aS-rotamers. The chiroptical properties of these axially chiral biphenol ethers in polar and nonpolar solvents were determined experimentally and were compared with the corresponding theoretical values to determine their conformational behavior in solution. Geometry optimization at the DFT-D/TZV2P level and subsequent time-dependent density functional theory (TD-DFT) at the BH-LYP/TZV2P level treatments to obtain rotatory strengths revealed that 6 out of 18 conformers (aR-Tg-, aR-Tg+, aR-G+t, aS-Tg-, aS-Tg+, and aS-G+t) are crucial to reproduce the experimental circular dichroism (CD) spectra and optical rotations. Although biphenyl molecules are in conformational equilibrium with varying interplanar angles in solution, our static approach to the prediction of the experimental CD spectra is simply based on pairs of thermally populated, local-minimum structures, that is, the dynamic behavior of the systems or the vibrational wave functions are not considered. The relative energies computed at the SCS-MP2/TZVPP level in the gas phase or in acetonitrile solution using the conductor-like screening model (COSMO) were found to be accurate enough to calculate the thermal population of the relevant conformers. Although most of the CD signals mutually cancel out each other between a pair of aR- and aS-rotamers, the remaining Cotton effects due to a small preference for a single rotamer produce characteristic CD spectra. In general (and somewhat unexpectedly), the delicate cancellation effects in the CD spectra are accurately described by the theoretical approach, and the simulated CD spectra are in excellent agreement with the experimental ones though observed rotatory strengths being always smaller (by 5-20 times) than the theoretical data. Accordingly, slight preferences for the P (or aR)-configuration for Biph22 and M-configuration for Biph33 and Biph44 are determined. The preference for the opposite isomer in the case of Biph22 is due to larger attractive intramolecular interactions between the chiral alkyl groups. This is also consistent with the lower oxidation potential found for Biph22 (DeltaE approximately 0.1 V), as compared with those for Biph33 and Biph44. The CT complex formation of Biph22-44 with various acceptors was also studied by UV-vis and CD spectroscopic methods.

Circular dichroism spectra demonstrate formation of the thrombin-binding DNA aptamer G-quadruplex under stabilizing-cation-deficient conditions

It is noteworthy that the formation of the DNA G-quadruplex is induced by factors other than stabilizing cations because this event probably occurs in living cells. Previous studies have shown that thrombin-binding DNA aptamer (TBA) forms a chair-type intramolecular G-quadruplex structure that binds with thrombin protein in the absence of stabilizing cations. Here, we used circular dichroism (CD) spectroscopy to confirm G-quadruplex formation in the presence of thrombin without stabilizing cations. We obtained characteristic CD spectra that demonstrated that TBA forms the distinctive G-quadruplex structure. Additionally, we investigated G-quadruplex formation induced by change of solvent environment: the influence of low-temperature conditions and molecular crowding.

IA3, an aspartic proteinase inhibitor from Saccharomyces cerevisiae, is intrinsically unstructured in solution.

IA(3) is a highly specific and potent 68-amino acid endogenous inhibitor of yeast proteinase A (YprA), and X-ray crystallographic studies have shown that IA(3) binds to YprA as an alpha-helix [Li, M., Phylip, L. H., Lees, W. E., Winther, J. R., Dunn, B. M., Wlodawer, A., Kay, J., and Gustchina, A. (2000) Nat. Struct. Biol. 7, 113-117]. Surprisingly, only residues 2-32 of IA(3) are seen in the X-ray structure, and the remaining residues are believed to be disordered in the complex. We have used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to show that IA(3) is unstructured in the absence of YprA. Specifically, IA(3) produced a CD spectrum characteristic of an unstructured peptide, and the (15)N HSQC NMR spectra of IA(3) were characteristic of a polypeptide lacking intrinsic structure. We characterized the unstructured state of IA(3) by using singular-value decomposition (SVD) to analyze the CD data in the presence of TFE, by fully assigning the unbound IA(3) protein by NMR and comparing the chemical shifts to published random-coil values, and by measuring (1)H-(15)N heteronuclear NOEs, which are all consistent with an unfolded protein. The IA(3) samples used for NMR analyses were active and inhibited YprA with an inhibition constant (K(i)) of 1.7 nM, and the addition of YprA led to a large spectral transition in IA(3). Calorimetric (ITC) data also show that the overall enthalpy of the interaction between IA(3) and YprA is exothermic.

DNA lesions derived from the site selective oxidation of Guanine by carbonate radical anions.

Carbonate radical anions are potentially important oxidants of nucleic acids in physiological environments. However, the mechanisms of action are poorly understood, and the end products of oxidation of DNA by carbonate radicals have not been characterized. These oxidation pathways were explored in this work, starting from the laser pulse-induced generation of the primary radical species to the identification of the stable oxidative modifications (lesions). The cascade of events was initiated by utilizing 308 nm XeCl excimer laser pulses to generate carbonate radical anions on submicrosecond time scales. This laser flash photolysis method involved the photodissociation of persulfate to sulfate radical anions and the one electron oxidation of bicarbonate anions by the sulfate radicals to yield the carbonate radical anions. The latter were monitored by their characteristic transient absorption band at 600 nm. The rate constants of reactions of carbonate radicals with oligonucleotides increase in the ascending order: 5'-d(CCATCCTACC) [(5.7 +/- 0.6) x 10(6) M(-)(1) s(-)(1)] < 5'-d(TATAACGTTATA), self-complementary duplex [(1.4 +/- 0.2) x 10(7) M(-)(1) s(-)(1)] < 5'-d(CCATCGCTACC [(2.4 +/- 0.3) x 10(7) M(-)(1) s(-)(1)] < 5'-d(CCATC[8-oxo-G]CTACC) [(3.2 +/- 0.4) x 10(8) M(-)(1) s(-)(1)], where 8-oxo-G is 8-oxo-7,8-dihydroguanine, the product of a two electron oxidation of guanine. This remarkable enhancement of the rate constants is correlated with the presence of either G or 8-oxo-G bases in the oligonucleotides. The rate constant for the oxidation of G in a single-stranded oligonuclotide is faster by a factor of approximately 2 than in the double-stranded form. The site selective oxidation of G and 8-oxo-G residues by carbonate radicals results in the formation of unique end products, the diastereomeric spiroiminodihydantoin (Sp) lesions, the products of a four electron oxidation of guanine. These lesions, formed in high yields (40-60%), were isolated by reversed phase HPLC and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These assignments were supported by the characteristic circular dichroism spectra of opposite signs of the two lesions. The oxidation of guanine to Sp diastereomers occurs, at least in part, via the formation of 8-oxo-G lesions as intermediates. The Sp lesions can be considered as the terminal products of the oxidation of G and 8-oxo-G in DNA by carbonate radical anions. The mechanistic aspects and biological implications of these site selective reactions in DNA initiated by carbonate radicals are discussed.

CHARACTERIZATION OF PHYTOCHELATIN-LIKE COMPLEXES FROM FLAX (LINUM USITATISSIMUM) SEED

Proteins were extracted from dehulled and delipidated powder of flaxseed (cv. NorMan), and fractionated by DEAE-Sephacel anion exchange chromatography. Ultraviolet absorbance scans of ion exchange fractions eluting in buffers at pH 8.6 containing 0. 45 M and 0.50M NaCl revealed A 255 >A 280 . Furthermore, the A 255 was reduced upon acidification and restored by adjusting to the original pH, confirming spectral characteristics typical of metal-thiolate complexes. Characteristic circular dichroism spectra and reversible aggregation-dissociation behavior under low and high salt conditions observed for these fractions are also typical of metal binding phytochelatins reported for other plants, while the presence of aromatic amino acids indicated by fluorescence spectra suggests some similarity to nonmetallothionein proteins. Further work is underway to investigate the prevalence of these and other complexes as well as the distribution of metals in different protein fractions of flaxseed, as a function of cultivar and geographical location.

Circular dichroism spectroscopy reveals invariant conformation of guanine runs in DNA.

We demonstrate that the characteristic circular dichroism (CD) features of the parallel-stranded DNA tetraplex of d(G4), especially the strong band at 260 nm, are characteristic for the B and A forms of the antiparallel duplex of d(C4G4). Hence, this band evidently originates from intrastrand guanine-guanine stacking, which is therefore very similar in the duplex and tetraplex DNA. In addition, the same type of the CD spectrum is provided by the ordered single strand of d(GA)10. This observation suggests that the ordered single strand of d(GA)10 is stabilized by a core of guanines stacked like in the parallel tetraplex. This view is used to start the modeling of the molecular structure of the ordered d(GA)10 single strand. Our studies suggest that guanine itself is strong enough to stabilize various secondary structures of DNA, which is a property relevant to thinking about the origin and evolution of molecular replicators.