Enantiomeric 4‐Acylamino‐6‐alkyloxy‐2 Alkylthiopyrimidines As Potential A3 Adenosine Receptor Antagonists: HPLC Chiral Resolution and Absolute Configuration Assignment by a Full Set of Chiroptical Spectroscopy

The chiral separation of enantiomeric couples of three potential A₃ adenosine receptor antagonists: (R/S)‐N‐(6‐(1‐phenylethoxy)‐2‐(propylthio)pyrimidin‐4‐yl)acetamide ( 1 ), (R/S)‐N‐(2‐(1‐phenylethylthio)‐6‐propoxypyrimidin‐4‐yl)acetamide ( 2 ), and (R/S)‐N‐(2‐(benzylthio)‐6‐sec‐butoxypyrimidin‐4‐yl)acetamide ( 3 ) was achieved by high‐performance liquid chromatography (HPLC). Three types of chiroptical spectroscopies, namely, optical rotatory dispersion (ORD), electronic circular dichroism (ECD), and vibrational circular dichroism (VCD), were applied to enantiomeric compounds. Through comparison with Density Functional Theory (DFT) calculations, encompassing extensive conformational analysis, full assignment of the absolute configuration (AC) for the three sets of compounds was obtained. Chirality 28:434–440, 2016. © 2016 Wiley Periodicals, Inc.     

Absolute configuration of eremophilane sesquiterpenes from Petasites hybridus: Comparison of experimental and calculated circular dichroism spectra

In‐depth conformational analyses of 10 known eremophilane (= (1S,4aR,7R,8aR)‐decahydro‐1,8a‐dimethyl‐7‐(1‐methylethyl)napththalene) sesquiterpenes, 1 – 10 , from Petasites hybridus were performed with molecular mechanics as well as density functional theory methods. Electronic transition energies and rotational strengths of these eight eremophilane lactones and two petasins were calculated by time‐dependent density functional theory (B3PW91/TZVP). The absolute configurations of the constituents could be assigned by comparison of their simulated and experimental circular dichroism (CD) spectra in methanol as (4S,5R,8S,10R) ( 1 , 2 ), (2R,4S,5R,8S,10R) ( 3 , 4 , 5 ), (2R,4S,5R,8R,9R,10R) ( 6 ), (2R,4S,5R,8R,10R) ( 7 , 8 ), and (3R,4R,5R) ( 9 , 10 ). Single‐crystal X‐ray diffraction data of 8β‐hydroxyeremophilanolide ((8S)‐8‐hydroxyeremophil‐7(11)‐en‐12,8‐olide) ( 1 ) served as starting point for the theoretical conformational calculations of the 8β‐epimers of the eremophilane lactones. Experimental CD spectra as well as ¹H NMR spectra of compound 1 in methanol were considerably dependent on sample concentration. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Isolation of the major chiral compounds from Bubonium graveolens essential oil by HPLC and absolute configuration determination by VCD

The chirality issues in the essential oils (EOs) of leaves and flowers from Bubonium graveolens were addressed by chiral high‐performance liquid chromatography (HPLC) with polarimetric detection and vibrational circular dichroism (VCD). The chemical compositions of the crude oils of three samples were established by gas chromatography / mass spectrometry (GC/MS). The well‐known cis ‐chrysanthenyl acetate ( 1 ), oxocyclonerolidol ( 2 ), and the recently disclosed cis ‐acetyloxychrysanthenyl acetate ( 3 ), the three major chiral compounds, were isolated by preparative HPLC. The naturally occurring oxocycloneroledol ( 2 ), mostly found in the leaf oil (49.4–55.6%), presents a (+) sign in the mobile phase during HPLC on a chiral stationary phase (CSP) with a Jasco polarimetric detection. The naturally occurring cis ‐chrysanthenyl acetate ( 1 ) and cis ‐acetyloxychrysanthenyl acetate ( 3 ), mostly found in the flower EO (35.9–74.9% and 10.0–34.3%, respectively), both present a (−) sign. HPLC on a CSP with polarimetric detection is an unprecedented approach to readily differentiate the flower and leaf EOs according to their chiral signature. The comparison of the experimental and calculated VCD spectra of pure isolated 1 , 2, and 3 provided their absolute configuration as being (1S ,5R ,6S )‐(−)‐2,7,7‐trimethylbicyclo[3.1.1]hept‐2‐en‐6‐yl acetate 1 , (2R ,6R )‐(+)‐6‐ethenyl‐2,6‐dimethyl‐2‐(4‐methylpent‐3‐en‐1‐yl)dihydro‐2H‐pyran‐3(4H)‐one) 2 and (1S ,5R ,6R ,7S )‐(−)‐7‐(acetyloxy)‐2,6‐dimethylbicyclo[3.1.1]hept‐2‐en‐6‐yl]methyl acetate 3 . Compounds 1 , 2, and 3 were already known in B. graveolens but this is the first report of the absolute configuration of (+)‐ 2 and (−)‐ 3 . The VCD chiral signatures of the crude oils were also recorded.     

Cis-diastereoselectivity in pictet-spengler reactions of L-tryptophan and electronic circular dichroism studies

The diastereoselective synthesis of optically active 1,3‐disubstituted tetrahydro‐β‐carbolines using polar protic Pictet–Spengler cyclization of (S)‐tryptophan methyl ester with five aldehydes RCHO (R═CH₃, C₂H₅, C₃H₇, C₄H₉, and C₆H₅) was studied. As an alternate route, the cyclization of (S)‐tryptophan with the same aldehydes and subsequent methylation of the resulting tetrahydro‐β‐carboline carboxylic acids were also performed for comparison. ¹³C NMR and electronic circular dichroism (ECD) studies and time‐dependent density functional theory ECD calculations data established the relative 1,3 cis/trans and the absolute configuration (1S,3S/ 1R,3S) of the synthesized compounds. The solid‐state and solution ECD study of the prepared compounds, supported by ECD calculation and X‐ray data, afforded a reliable ECD method for the configurational assignment of 1,3‐disubstituted tetrahydro‐β‐carbolines and revealed the stereochemical factors that determine the characteristic ECD data. Chirality 24:789–795, 2012. © 2012 Wiley Periodicals, Inc.     

Reliable HPLC separation,vibrational circular dichroism spectra,and absolute configurations of isoborneol enantiomers

Resolution of chiral compounds has played an important role in the pharmaceutical field, involving detailed studies of pharmacokinetics, physiological, toxicological, and metabolic activities of enantiomers. Herein, a reliable method by high‐performance liquid chromatography (HPLC) coupled with an optical rotation detector was developed to separate isoborneol enantiomers. A cellulose tris(3, 5‐dimethylphenylcarbamate)‐coated chiral stationary phase showed the best separation performance for isoborneol enantiomers in the normal phase among four polysaccharide chiral packings. The effects of alcoholic modifiers and column temperature were studied in detail. Resolution of the isoborneol racemate displayed a downward trend along with an increase in the content of ethanol and column temperature, indicating that less ethanol in the mobile phase and lower temperature were favorable to this process. Moreover, two isoborneol enantiomers were obtained via a semipreparative chiral HPLC technique under optimum conditions, and further characterized by analytical HPLC, and experimental and calculated vibrational circular dichroism (VCD) spectroscopy, respectively. The solution VCD spectrum of the first‐eluted component was consistent with the Density Functional Theory (DFT) calculated pattern based on the SSS configuration, indicating that this enantiomer should be (1S , 2S , 4S )‐(+)‐isoborneol. Briefly, these results have provided reliable information to establish a method for analysis, preparative separation, and absolute configuration of chiral compounds without typical chromophoric groups.     

Absolute configuration of eremophilanoids by vibrational circular dichroism

The absolute configurations (AC) of natural occurring 6-hydroxyeuryopsin (1), of its acetyl derivative 2, and of eremophilanolide 8 were confirmed by comparison of the experimental vibrational circular dichroism (VCD) spectra with theoretical curves generated from density functional theory (DFT) calculations. Initial analyses were carried out using a Monte Carlo searching with the MMFF94 molecular mechanics force field. All MMFF94 conformers were further optimized using DFT at the B3LYP/6-31G(d) level of theory, followed by calculations of their vibrational frequencies at the B3LYP/6-31G(d,p); the VCD spectra of 2 and 8 were also calculated at the B3PW91/DGDZVP level of theory. Good agreement between theoretical and experimental VCD curves unambiguously verified the 4S,5R,6S absolute configuration for 1 and 2, and the 1S,4S,5R,6S,8S,10S configuration for 8.     

Determination of absolute configuration using vibrational circular dichroism spectroscopy: the chiral sulfoxide 1-thiochromanone S-oxide

We reexamined the absolute configuration (AC) of the chiral sulfoxide 1-thiochromanone S-oxide (1) using vibrational circular dichroism (VCD) spectroscopy. The VCD spectrum of 1 was analyzed using density functional theory (DFT). DFT predicts two stable conformations of 1, separated by <1 kcal/mole. Their VCD spectra were calculated using the DFT/GIAO methodology. The VCD spectrum predicted for the equilibrium mixture of the two conformations of (S)-1 is in excellent agreement with the experimental spectrum of (+)-1. The AC of 1 is therefore definitively R(-)/S(+).     

Absolute configuration of the ocimene monoterpenoids from Artemisia absinthium

The absolute configuration (AC) of the naturally occurring ocimenes (−)‐(3S ,5Z )‐2,6‐dimethyl‐2,3‐epoxyocta‐5,7‐diene ( 1 ) and (−)‐(3S ,5Z )‐2,6‐dimethylocta‐5,7‐dien‐2,3‐diol ( 2 ), isolated from the essential oils of domesticated specimens of Artemisia absinthium , followed by vibrational circular dichroism (VCD) studies of 1 , as well as from the acetonide 3 and the monoacetate 4 , both derived from 2 , since secondary alcohols are not the best functional groups to be present during VCD studies in solution due to intermolecular associations. The AC follows from comparison of experimental and calculated VCD spectra that were obtained by Density Functional Theory computation at the B3LYP/DGDZVP level of theory. Careful nuclear magnetic resonance (NMR) measurements were compared with literature values, providing for the first time systematic ¹H and ¹³C chemical shift data. Regarding homonuclear ¹H coupling constants, after performing a few irradiation experiments that showed the presence of several small long‐range interactions, the complete set of coupling constants for 3 , which is representative of the four studied molecules, was determined by iterations using the PERCH software. This procedure even allowed assigning the pro ‐R and pro ‐S methyl group signals of the two gem ‐dimethyl groups present in 3 .     

The effect of temperature and salt concentration on the circular dichroism exhibited by unionized derivatives of L-alanine in aqueous solution

The circular dichroism of Ac–Ala–NHMe, cyclo(–Ala–Ala–), Ac–Ala–OMe, Ac–Ala–Ala–OMe, and Ac–Ala–Ala–Ala–OMe has been measured in water and in aqueous salt solutions as a function of temperature. Only cyclo(–Ala–Ala–) exhibits circular dichroism which is independent of temperature. Each of the linear derivatives of L -alanine exhibits a positive circular dichroism in the range 208–218 nm at 15°C in water. Heating reduces the intensity of the positive circular dichroism, and only Ac–Ala–OMe retains positive circular dichroism at 75°C in water. Isothermal addition of salts produces changes in the circular dichroism of linear derivatives of L -alanine which resemble those seen on heating. The relative effectiveness of the salts tested, at a concentration of 4M, is LiCl ? KCl = NaCl < MgCl₂ ? CaCl₂ ? NaClO₄. The circular dichroism of cyclo(–Ala–Ala–) is also affected by the salts. Extrapolation of the results obtained with Ac–Ala–OMe, Ac–Ala–Ala–OMe, and Ac–Ala–Ala–Ala–OMe to a long polypeptide with a –CH₂R side chain in the L -configuration leads to the conclusion that this polypeptide should exhibit a temperature-dependent salt-sensitive positive circular dichroism between 208 and 218 nm when it exists as a statstical coil.     

Stereochemical assignment of fusiccocadiene from NMR shielding constants and vibrational circular dichroism spectroscopy

The absolute configuration (AC) of the common precursor of the fusicoccane family of terpenoids, fusicocca‐2,10(14)‐diene (FCdiene), had only been deduced by a lengthy total synthesis, or indirectly from crystal structures of fusicoccin A. However, in particular the AC determinations based on downstream products of the terpene synthase intrinsically overlook potential epimerization reactions. In this contribution, we confirm the relative stereochemistry of FCdiene by comparison of experimental and predicted ¹³C–NMR chemical shifts, and finally determine the absolute configuration from an analysis of its infrared and vibrational circular dichroism spectra.     

Chaperone SecB: Conformational changes demonstrated by circular dichroism

The chaperone SecB, which is involved in protein export inEscherichia coli, is shown by circular dichroism measurements to contain a high content of-pleated sheets. Prediction of the secondary structure of SecB is in good agreement with the observed content of-sheet. In accordance with the previous studies in which changes in conformation were assessed indirectly [Randall (1992),Science 257, 241–245], here we show that the conformation of SecB changes with the concentration of salt in the milieu and also when SecB interacts with a peptide ligand.Abbreviations ANS 1-anilino-naphthalene-8-sulfonate - CD circular dichroism - NMR nuclear magnetic resonance - CCA convex constraint analysis     

Enantioselective ene‐reduction of E‐2‐cyano‐3‐(furan‐2‐yl) acrylamide by marine and terrestrial fungi and absolute configuration of (R)‐2‐cyano‐3‐(furan‐2‐yl) propanamide determined by calculations of electronic circular dichroism (ECD) spectra

This work reports the green organic chemistry synthesis of E‐2‐cyano‐3(furan‐2‐yl) acrylamide under microwave radiation (55 W), as well as the use of filamentous marine and terrestrial‐derived fungi, in the first ene‐reduction of 2‐cyano‐3‐(furan‐2‐yl) acrylamide to (R)‐2‐cyano‐3‐(furan‐2‐yl)propanamide. The fungal strains screened included Penicillium citrinum CBMAI 1186, Trichoderma sp. CBMAI 932 and Aspergillus sydowii CBMAI 935, and the filamentous terrestrial fungi Aspergillus sp. FPZSP 146 and Aspergillus sp. FPZSP 152. A compound with an uncommon CN‐bearing stereogenic center at the α‐C position was obtained by enantioselective reactions mediated in the presence of the microorganisms yielding the (R)‐2‐cyano‐3‐(furan‐2‐yl) propanamide 3a . Its isolated yield and e.e. ranged from 86% to 98% and 39% to 99%, respectively. The absolute configuration of the biotransformation products was determined by time‐dependent density functional theory (TD‐DFT) calculations of electronic circular dichroism (ECD) spectra. Finally, the tautomerization of 2‐cyano‐3‐(furan‐2‐yl) propanamide 3a to form an achiral ketenimine was observed and investigated in presence of protic solvents.     

Biochemical and biophysical studies on cytochrome c oxidase. XVIII. Potentiometric titrations of cytochrome c oxidase followed by circular dichroism

1. Potentiometric circular dichroism titrations of cytochrome c oxidase, carried out in the absence of cytochrome c, confirm the potentiometric equivalence of the two heme a groups of cytochrome c oxidase. In the presence of cytochrome c, two different midpoint potentials are found for the two heme a groups of cytochrome c oxidase.2. Circular dichroism difference spectra (reduced minus oxidized) of the two heme a components of cytochrome c oxidase have been obtained by means of this potentiometric titration. On reduction of the first heme a group a circular dichroism difference spectrum is obtained with peaks at 425, 442 and 602.5 nm; the second heme a group shows difference peaks at 434, 447 and 608 nm. Whereas both heme a groups contribute about equally to the absorbance difference spectrum, the second heme a group reduced contributes about twice as much to the circular dichroism difference spectrum as does the first heme a group.3. From these spectral and circular dichroism differences it is concluded that, on reduction of or ligand binding to cytochrome c oxidase, conformational changes occur which affect the symmetry of the environments of the heme a groups.     

Protein secondary structure from circular dichroism spectra

Circular dichroism spectra of proteins are extremely sensitive to secondary structure. Nevertheless, circular dichroism spectra should not be analyzed for protein secondary structure unless they are measured to at least 184 nm. Even if all the various types ofβ-turns are lumped together, there are at least 5 different types of secondary structure in a protein (α-helix, antiparallelβ-sheet, parallelβ-sheet,β-turn, and other structures not included in the first 4 categories). It is not possible to solve for these 5 parameters unless there are 5 equations. Singular value decomposition can be used to show that circular dichroism spectra of proteins measured to 200 nm contain only 2 pieces of information, while spectra measured to 190 nm contain about 4. Adding the constraint that the sum of secondary structures must equal 1 provides another piece of information, but even with this constraint, spectra measured to 190 nm simply do not analyze well for the 5 unknowns in secondary structure. Spectra measured to 184 nm do contain 5 pieces of information and we have used such spectra successfully to analyze a variety of proteins for their component secondary structures.     

Discrimination of Axial and Central Stereogenic Elements in Chiral Bis(oxazolines) Based on Atropisomeric 3,3′‐Bithiophene Scaffolds Through Chiroptical Spectroscopies

Two diastereoisomeric pairs of bis‐oxazolines, provided with a stereogenic center at carbon 4 and based on the 3,3′‐bithiophene atropisomeric scaffold, were synthesized and structurally characterized. They differ in the substituents at positions 2 and 5 of the thiophene rings, which are functionalized with methyl (1) or phenyl (2) groups, respectively. In vibrational circular dichroism (VCD) spectra, recorded in CCl₄ solutions, it is possible to distinctly recognize the characteristic features of axial and central stereogenic elements. In tandem with Density Functional Theory (DFT) calculations, the absolute configuration (AC) of the diastereoisomers was safely established. In this case, VCD was shown to be superior to ECD (electronic circular dichroism) in the assignment of AC. The normal modes, evaluated from DFT calculations, show that the VCD signals in correspondence with the stereogenic axis of the bithiophene unit are different for 1 and 2. The VCD spectra of a molecular analog of 1, the (S)‐2,2′,5,5′‐tetramethyl‐4,4′‐bis‐(diphenylphosphino)‐3,3′‐bithiophene oxide (3), characterized by the same 3,3′‐bithiophene scaffold, but devoid of stereogenic centers, exhibits signals similar to those observed in the case of diastereoisomer (aS,R,R)‐1a, associated with almost identical normal modes. Chirality 28:686–695, 2016. © 2016 Wiley Periodicals, Inc.     

Solvent‐dependent inversion of circular dichroism signal in naproxen: An unusual effect!

The electronic circular dichroism (ECD) spectra of naproxen enantiomers were studied as a function of solvents using experimental (circular dichroism) and theoretical (time‐dependent density functional theory) approaches. The (R)‐ and (S)‐naproxen enantiomers presented an unusual inversion in their ECD signals in the presence of ethanol and water when compared with polar aprotic solvents such as acetonitrile. From a practical point of view, these findings deserve great attention because these solvents are widely used for high‐performance liquid chromatography analysis in quality control of chiral pharmaceutical drugs. This is particularly relevant to naproxen because the (S)‐naproxen has anti‐inflammatory properties, whereas (R)‐naproxen is hepatotoxic. A time‐dependent density functional theory computer simulation was conducted to investigate the signal inversion using the solvation model based on density, a reparameterization of polarized continuum model. Electronic circular dichroism signals of conformers were calculated by computer simulation and their contribution to the combined spectra obtained according to Boltzmann weighting. It was found that the experimentally observed ECD signal inversion can be associated with the minor or major contribution of different conformers of naproxen.     

Partial analysis of the bands seen in circular dichroism spectra of green and blue-green algal cells and thylakoids

A comparison was made of the circular dichroism (C.D.) spectra of Chlorella, Euglena, and Anacystis cells and thylakoids. Analyses of the spectra reveal that these C.D. bands are similar to those observed previously in whole spinach choloroplasts and subchloroplast particles. C.D. spectra of Euglena chloroplasts show bands at longer wavelengths than previously reported. From comparisons of circular dichroism spectra and fine structure, it was concluded that: (a) bands seen in circular dichroism spectra were not the result of light scattering from thylakoid membranes; and (b) bands seen in the C.D. spectra of nonmembranous systems (previously reported) could account for circular dichroism of algae. We also concluded that comparisons would have to be made with model systems in order to correct for effects of absorption flattening, concentration obscuring, and differential light scattering of membranous systems.     

Synthesis,Characterization and Evaluation of Cytotoxic Activities of Novel Aziridinyl Phosphonic Acid Derivatives

New aziridine 2‐phosphonic acids were prepared by monohydrolysis of the aziridine 2‐phosphonates that were obtained by the modified Gabriel?Cromwell reaction of vinyl phosphonate or α‐tosylvinyl phosphonate with a primary amine or a chiral amine. The cellular cytotoxicity of these compounds was tested against the HCT‐116 colorectal cancer cell lines and the CCD‐18Co normal colon fibroblast lines using the MTT assay. Three of the synthesized phosphonic acid derivatives 2e (ethyl hydrogen {(2S)‐1‐[(1S)‐1‐(naphthalen‐2‐yl)ethyl]aziridin‐2‐yl}phosphonate), 2h (ethyl hydrogen (1‐benzylaziridin‐2‐yl)phosphonate), and 2i (ethyl hydrogen (1‐cyclohexylaziridin‐2‐yl)phosphonate) showed higher cytotoxicity than the reference cancer treatment agent etoposide. Cell death was through a robust induction of apoptosis even more effectively than etoposide, a well‐known apoptosis inducing agent.     

Absolute configuration of labdanes and ent-clerodanes from Chromolaena pulchella by vibrational circular dichroism

The aerial parts of Chromolaena pulchella biosynthesize two groups of diterpenes belonging to opposite enantiomeric series, specifically, the furanoid ent-clerodanes (5R,8R,9S,10R)-(-)-hardwikiic acid (1), methyl (5R,8R,9S,10R)-(-)-hardwikiate (2), (5S,8R,9S,10R)-(-)-hautriwaic acid lactone (3), methyl (5R,8R,9S,10R)-(-)-nidoresedate (4) and methyl (8R,9R)-(-)-strictate (5), as well as the labdanes (5S,8R,9R,10S)-(+)-(13E)-labd-13-ene-8,15-diol (6) and (5S,8R,9R,10S)-(+)-isoabienol (7). The absolute configuration of the two groups of diterpenes was unambiguously assigned by comparison of the vibrational circular dichroism spectra of 3 for ent-clerodanes, and of 7 for labdanes with their theoretical spectra obtained by density functional theory calculations. The results support a biogenetic proposal to diterpenes found in the studied botanical species.     

Determination of the absolute configurations of natural products via density functional theory calculations of vibrational circular dichroism, electronic circular dichroism, and optical rotation: the iso-schizozygane alkaloids isoschizogaline and isoschizogamine

The development of density functional theory (DFT) methods for the calculation of vibrational circular dichroism (VCD), electronic circular dichroism (ECD), and transparent spectral region optical rotation (OR) has revolutionized the determination of the absolute configurations (ACs) of chiral molecules using these chiroptical properties. We report the concerted application of DFT calculations of VCD, ECD, and OR to the determination of the ACs of the isoschizozygane alkaloid natural products, isoschizogaline, and isochizogamine, whose ACs have not previously been determined. The ACs of naturally occurring (-)-isoschizogaline and (-)-isoschizogamine, are both determined definitively to be 2R, 7R, 20S, 21S.