Chiral organic stereochemistry: Chiral HPLC, chiral auxiliaries, CD spectroscopy, X-ray crystallography, and light-powered chiral molecular motors.

In the studies of chiral organic stereochemistry, it is important to use enantiopure compounds. For this purpose, the chiral HPLC (High-Pressure Liquid Chromatography) columns containing chiral stationary phases were invented by Y. Okamoto and coworkers for enantio-separating various racemic compounds. In addition, the use of chiral auxiliaries is also useful for preparing enantiopure compounds and also for determining their absolute configurations, where covalent-bonded diastereomers are separated by HPLC on silica gel. In this review article, these HPLC methods will be discussed together with the applications to some interesting organic compounds including light-powered chiral molecular motors.

Exploration of chromophores for a VCD couplet in a spectrally transparent infrared region for biomolecules.

Interactions of two chromophores such as carbonyl groups yield a strong VCD couplet that reflects the molecular structures. The use of VCD couplets for biomacromolecular structural studies has been hampered by severe signal overlap caused by numerous functional groups that originally exist in biomacromolecules. Nitrile, isonitrile, alkyne, and azido groups show characteristic IR absorption in the 2300-2000 cm-1 region, where biomolecules do not strongly absorb. We herein examined the usefulness of these functional groups as chromophores to observe a strong VCD couplet that can be readily interpreted using theoretical calculations. Studies on a chiral binaphthyl scaffold possessing two identical chromophores showed that nitrile and isonitrile groups generate moderately-strong but complex VCD signals due to anharmonic contributions. The nature of their anharmonic VCD patterns is discussed by comparison with the VCD spectrum of a mono-chromophoric molecule and by anharmonic DFT calculations. On the other hand, through studies on diazido binaphthyl and diazido monosaccharide, we demonstrated that the azido group is more promising for structural analysis of larger molecules due to its simple, strong VCD couplet whose spectral patterns are readily predicted by harmonic DFT calculations.

The enduring legacy of Koji Nakanishi's research on natural products and bioorganic chemistry. Part 2. Inception and establishment of the ECD exciton chirality method in 1960s to 1970s: A marvel of Nakanishi's Japanese team.

The electronic circular dichroism (ECD) exciton chirality method is very useful for determining the absolute configuration (AC) of chiral compounds. In the ECD spectroscopy, the chromophore-chromophore interaction, ie, exciton coupling, is very important. For example, Harada and Nakanishi first discovered in 1969 that chiral dibenzoates exhibit exciton split bisignate Cotton effects, from the sign of which the screw sense between two long axes of benzoate chromophores, ie, the AC of dibenzoate, can be determined. This method was named the dibenzoate chirality rule and has been successfully applied to various natural products to determine their ACs. During these studies, it was also found that this CD method was expanded to encompass other aromatic and olefin chromophores like naphthalene, diene, enone, etc. Therefore, the name of the dibenzaote chirality rule was changed to the CD exciton chirality method. In 1970s, there were heated controversies about the inconsistency between X-ray Bijvoet and CD exciton chirality methods, which was a shocking and serious problem in the community of molecular chirality research. Harada and coworkers synthesized the most ideal cage compound with two anthracene chromophores to connect X-ray Bijvoet and CD exciton chitality methods and proved that these two methods are consistent with each other.

Thermal E/ Z Isomerization in First Generation Molecular Motors.

Determination of a thermal E/ Z isomerization barrier of first generation molecular motors is reported. Stable ( E)-1a directly converts to stable ( Z)-1c without photochemical E/ Z isomerization. The activation Gibbs energy of the isomerization was determined to be 123 kJ mol-1 by circular dichroism spectral changes. Density functional theory calculations show that ( Z)-1c is ∼11.4 kJ mol-1 more stable than ( E)-1a.

Chiral Molecular Science: How were the absolute configurations of chiral molecules determined? "Experimental results and theories".

Molecular chirality is a key concept in chemistry, bioscience, and molecular technology, like the invention of a light-powered chiral molecular motor explained in this review. Thus, the primary research subject is how to determine the absolute configuration (AC) of chiral compounds. This review article focuses on the principle, theory, and practice of the nonempirical methods for determining ACs of chiral compounds, i.e., the Bijvoet method in X-ray crystallography and the circular dichroism (CD) exciton chirality method, together with the historical aspects of AC determination. The theoretical equations of X-ray crystallography and exciton CD spectroscopy are explained in detail, and these equations are useful for readers to understand the principle and mechanism of these methods. This review also focuses on the relative methods, where the internal reference with known AC is used and the relative configuration is determined by X-ray crystallography and/or 1 H nuclear magnetic resonance (NMR) diamagnetic anisotropy method. In these cases, CSDP acid and MαNP acid are useful for the chiral resolution of racemic alcohols, where their diastereomeric esters are easily separable by high-performance liquid chromatography (HPLC) on silica gel. Thus, these methods are useful for the preparation of enantiopure compounds and simultaneous determination of their ACs. In this review article, the above methods are explained mainly based on the author's own research results.

HPLC Separation of Diastereomers: Chiral Molecular Tools Useful for the Preparation of Enantiopure Compounds and Simultaneous Determination of Their Absolute Configurations.

To obtain enantiopure compounds, the so-called chiral high performance liquid chromatography (HPLC) method, i.e., HPLC using a chiral stationary phase, is very useful, as reviewed in the present Special Issue. On the other hand, normal HPLC (on silica gel) separation of diastereomers is also useful for the preparation of enantiopure compounds and also for the simultaneous determination of their absolute configurations (ACs). The author and coworkers have developed some chiral molecular tools, e.g., camphorsultam dichlorophthalic acid (CSDP acid), 2-methoxy-2-(1-naphthyl)propionic acid (MαNP acid), and others suitable for this purpose. For example, a racemic alcohol is esterified with (S)-(+)-MαNP acid, yielding diastereomeric esters, which are easily separable by HPLC on silica gel. The ACs of the obtained enantiopure MαNP esters can be determined by the ¹H-NMR diamagnetic anisotropy method. In addition, MαNP or CSDP esters have a high probability of giving single crystals suitable for X-ray crystallography. From the X-ray Oak Ridge thermal ellipsoid plot (ORTEP) drawing, the AC of the alcohol part can be unambiguously determined because the AC of the acid part is already known. The hydrolysis of MαNP or CSDP esters yields enantiopure alcohols with the established ACs. The mechanism and application examples of these methods are explained.

[Comparison of tests results between T-SPOT.TB and QuantiFERON-TB Gold In-Tube in a contact investigation].

BACKGROUND: We compared T-SPOT.TB (T-SPOT) and QuantiFERON-TB Gold In-Tube (QFT-GIT) test results in a contact investigation. SUBJECTS AND METHODS: The index case was a male lecturer at a vocational school in Tokyo. Chest X-ray examinations and T-SPOT tests were performed on all 397 contacts, and QFT-GIT was performed on a subset of these contact subjects. RESULTS: Chest X-ray examination showed no evidence of tuberculosis in any subjects. Among 389 contacts that underwent T-SPOT testing, 5 showed a positive reaction, 3 showed borderline reactions (1 positive borderline and 2 negative borderline), and 381 were negative. Among 56 contacts tested using both QFT-GIT and T-SPOT, 4 were positive, 1 was borderline, and 51 were negative by QFT-GIT. By T-SPOT, 2 contacts were positive, 1 was borderline positive, and 53 were negative. Preventive chemotherapy was indicated for the 5 positive and 1 borderline positive contacts identified by the T-SPOT test. DISCUSSION: Chest X-ray examination and the T-SPOT test did not identify the TB outbreak. CONCLUSION: The majority of contact subjects were negative by both tests, suggesting that both have a high specificity in contact investigations. However, the moderate concordance rate indicates that further testing is necessary to fully evaluate these tests.

[Contact investigation using QuantiFERON-TB Gold test to evaluate TB exposure in 61 subjects in a hospital setting--(2) Change in QuantiFERON response during one year after exposure].

UNLABELLED: OBJECTIVES & SUBJECTS: The change in IGRA (interferon-gamma release assay, with QuantiFERON-TB Gold, QFT) responses was followed up for one year in a group of contacts of healthcare workers who had been exposed to tuberculosis (TB) infection for a relatively short period in a hospital. The observation was made of a total of 59 close contacts of the index case, where 16 showed positive QFT-conversion and 7 showed the intermediate response ranging 0.1 to 0.35 IU/mL. Three of the conversion cases developed active TB. RESULTS: 67% of the QFT conversions occurred within 2 months of exposure and the others between 2 to 9 months. Those having converted later than 2 months after the exposure showed generally weaker QFT responses than the earlier converters. In response to the treatment to converters (either to latent TB infection or to active TB), 80% of the cases reversed to negative or intermediate. The geometric means of the response values for ESAT-6 and CFP-10 also showed significant decline over the treatment time. DISCUSSIONS: The time profile of responses in the intermediate responders revealed an obviously distinct pattern from that of the negative responders with the values remaining uniformly at very low level throughout, which suggests that this group includes somehow exceptional responders either with or without infection.

Surface modification of cellulose with succinic anhydride in dimethyl sulfoxide using potassium carbonate as a catalyst.

Cellulose succinates (CSs) having degrees of substitution (DSs) ranging from 0.78 to 2.77 were successfully obtained by reacting cellulose with succinic anhydride (SA) in dimethyl sulfoxide at room temperature using a small amount of inexpensive solid potassium carbonate as a catalyst. Interestingly, CSs with higher DS values were obtained with a much smaller amount of catalyst than previously reported. Moreover, it is possible to control the DS by tailoring the reaction time and mass ratio of cellulose/SA. The hydroxyl groups at the C-6, C-2, and C-3 positions were the main esterification positions. In this process, most of the raw materials are either incorporated into the product or are recoverable. The E-factor, which reflects the sustainability of a given process, was demonstrated to be reduced by 93% by recovering the raw materials.

Transient role of CD4+CD25+ regulatory T cells in mycobacterial infection in mice.

CD4(+)CD25(+) regulatory T (Treg) cells cause immune suppression by inhibiting T cell effector functions and play pivotal roles not only in self-tolerance but also in immune response to parasitic microbial pathogens. Mycobacteria are major parasitic bacterial pathogens, but the role of CD4(+)CD25(+) Treg cells in mycobacterial infection is not yet defined. In this study we found that, at the early stage of infection, depletion of CD25(+) cells reduced both bacterial load and granuloma formation in mice infected with Mycobacterium tuberculosis strains, such as M. tuberculosis Erdman or M. tuberculosis Kurono. However, at a later stage of infection, bacterial burden and histopathology were similar regardless of depletion of CD25(+) cells. Severe combined immunodeficient (SCID) mice reconstituted with CD4(+)CD25(-) T cells alone or a combination of CD4(+)CD25(+) and CD4(+)CD25(-) T cells showed similar bacterial loads and survival kinetics after infection with M. tuberculosis Erdman. Consistent with in vivo data, in vitro studies revealed that mycobacterial antigens, purified protein derivative of tuberculin (PPD), failed to induce the suppressive function of CD4(+)CD25(+) Treg cells to CD4(+)CD25(-) effector T cells, as demonstrated by the lack of response of CD4(+)CD25(+) T cells to PPD, in mice chronically infected with Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis. Our data show that CD4(+)CD25(+) Treg cells have a transient effect at the early stage of mycobacterial infection but, contrary to the expectation, have little impact on the overall course of infection.

Performance of a Whole-Blood Interferon-Gamma Release Assay with Mycobacterium RD1-Specific Antigens among HIV-Infected Persons.

OBJECTIVE: To evaluate the usefulness of one of IGRAs, QuantiFERON-TB Gold (QFT-G), in human immunodeficiency virus- (HIV-) infected patients with various CD4(+) T cell counts. METHODS: The QFT-G assay was performed using QFT-G kits among 107 HIV-infected patients including 9 cases with active tuberculosis (TB). RESULTS: In HIV-infected patients with CD4(+) > 50/microL, QFT-G positive rate for active TB patients was 5/6 (sensitivity = 83%), and that for those without active disease was 1/69 (specificity = 99%). The frequency of indeterminate QFT-G test was significantly higher in those with CD4(+) less than 50/microL (P < .0001). At the same time there was a proportional relationship between CD4(+) and interferon-gamma response to mitogen (positive control) in QFT-G test (P = .0001). Conclusions. Our data suggested that QFT-G had high sensitivity and specificity in HIV-infected populations with CD4(+) greater than 50/microL. However, QFT-G did not perform well in HIV-positive patients with CD4(+) less than 50/microL.

[A view of dynamics of immune responses against tuberculosis infection through QFT].

QFT has been approved as a diagnostic test of TB infection in April 2005 in Japan, and further adopted to health insurance in January 2006. QFT is now a necessary tool to diagnose TB infection, especially in contact investigation. Since QFT uses M. tuberculosis-specific antigens, which are absent from BCG and most of non-tuberculous mycobacteria, to stimulate blood samples, and Interferon-gamma (IFN-gamma) produced with antigen-specific T cells is measured to diagnose TB infection, QFT can specifically diagnose TB infection without influence of BCG vaccination or infection of most of non-tuberculous mycobacteria. There is another diagnostic test, T-SPOT.TB, which uses similar antigens to QFT, and these two tests are called as IGRAs (Interferon-Gamma Release Assays). However, as production of IFN-gamma is a small part of protective immune responses against TB infection, it is difficult from this point of view to understand the dynamics of protective immune responses against TB infection through IGRAs results. Especially, it is impossible to distinguish between active TB and latent TB infection, to identify time of TB infection, or to detect dormancy TB infection using current IGRAs. Using biomarkers other than IFN-gamma or antigens other than antigens used in current IGRAs, development of newer diagnostic tests which have these performances would be awaited.

Penetration of Singlet Oxygen into Films with Oxygen Permeability Coefficient Close to that of Skin.

Although its antiviral and antibacterial functions help prevent infection, singlet oxygen (1 O2 )-which is generated by the action of light on an endogenous photosensitizer-is cytotoxic. In the present study, we investigated the ability of 1 O2 -generated by the action of visible light on a photosensitizer-to penetrate skin. We used two polymer films with oxygen permeability coefficients similar to that of skin-i.e. cellulose acetate (CA) and ethyl cellulose (EC). Both films contained 1,3-diphenylisobenzofuran (DPBF), which was used as an 1 O2 probe. 1 O2 generated externally did not permeate the films by mere contact. Therefore, we conclude that the potential for 1 O2 to penetrate the skin is very low, and films that generate 1 O2 are safe and useful for preventing infections by contact. We also proved that 1 O2 can move between the layers of integrated polymer films when they are joined together.

Enantiomerically pure alleno-acetylenic macrocycles: synthesis, solid-state structures, chiroptical properties, and electron localization function analysis.

New enantiomerically pure alleno-acetylenic macrocycles were prepared by oxidative homocoupling of optically active 1,3-diethynylallenes. Enantiomer separation resulted from a combined strategy of synthesis and chiral HPLC techniques. Two other achiral stereoisomers were also isolated and fully characterized. In addition, the X-ray structures of the chiral D(4)- and C(2)-symmetric macrocycles are reported. The chiroptical properties of these macrocycles are discussed on the basis of quantum chemical calculations, by using the CAM-B3LYP functional. Studies were carried out to investigate the vibronic fine structure observed experimentally in the UV/Vis and CD spectra. The origin of the intense chiroptical response of the chiral alleno-acetylenic macrocycles is explained by considering the topology of the molecular orbitals involved, thus relating electronic properties to structural features. Further analysis of the canonical molecular orbitals and the electron localization function (ELF) shows that these macrocycles belong to a relatively rare class of highly stable and formally anti-aromatic Hückel compounds.

ECD cotton effect approximated by the Gaussian curve and other methods.

To determine the absolute configuration of chiral compounds, theoretical calculations of ECD spectra have been extensively carried out, where the shape of component CD Cotton effects was approximated by the Gaussian distribution curve. However, in some articles, errors are found in the equations of Gaussian curve expressing CD Cotton effects. In this short review, the correct and general forms of the Gaussian equation and the approximation of the CD curve by other methods are described.

[Interpretation of QFT results in several contact investigations].

QuantiFERON TB-2G (QFT-2G) is now widely used for contact investigations, as QFT-2G is not affected by BCG vaccination, whereas the conventional tuberculin skin test (TST) is confounded by BCG vaccination. We applied the QFT-2G tests to numerous contact investigations and found that the majority of contacts who had been supposed to be infected with M. tuberculosis (Mtb) based on TST were negative in the QFT-2G tests, strongly suggesting the possibility that the unnecessary preventive chemotheraphy was indicated for many TST positive contacts due to BCG vaccination in conventional contact investigations. Although QFT-2G positive results implicate that Mtb in certain active phases presents in the body, when Mtb had been infected cannot be decided by QFT-2G. Therefore, QFT-2G results should be carefully interpreted, especially in elder persons who had been exposed to Mtb in the past in Japan. Furthermore, since there were some active TB cases in QFT-2G negative contacts, attention must be paid for QFT-2G negative contacts in highly exposed groups. In this paper, I will discuss about current usages of QFT-2G based on our experiences of contact investigations.

Quality assessment of an interferon-gamma release assay for tuberculosis infection in a resource-limited setting.

BACKGROUND: When a test for diagnosis of infectious diseases is introduced in a resource-limited setting, monitoring quality is a major concern. An optimized design of experiment and statistical models are required for this assessment. METHODS: Interferon-gamma release assay to detect tuberculosis (TB) infection from whole blood was tested in Hanoi, Viet Nam. Balanced incomplete block design (BIBD) was planned and fixed-effect models with heterogeneous error variance were used for analysis. In the first trial, the whole blood from 12 donors was incubated with nil, TB-specific antigens or mitogen. In 72 measurements, two laboratory members exchanged their roles in harvesting plasma and testing for interferon-gamma release using enzyme linked immunosorbent assay (ELISA) technique. After intervention including checkup of all steps and standard operation procedures, the second trial was implemented in a similar manner. RESULTS: The lack of precision in the first trial was clearly demonstrated. Large within-individual error was significantly affected by both harvester and ELISA operator, indicating that both of the steps had problems. After the intervention, overall within-individual error was significantly reduced (P < 0.0001) and error variance was no longer affected by laboratory personnel in charge, indicating that a marked improvement could be objectively observed. CONCLUSION: BIBD and analysis of fixed-effect models with heterogeneous variance are suitable and useful for objective and individualized assessment of proficiency in a multistep diagnostic test for infectious diseases in a resource-constrained laboratory. The action plan based on our findings would be worth considering when monitoring for internal quality control is difficult on site.

Comparing various chiral dirhodium tetracarboxylates in the dirhodium method.

Three enantiopure dirhodium tetracarboxylates are compared in their NMR properties to differentiate chiral ligands of various kinds (dirhodium method). The complex with four (S)-2-methoxy-2-(1-naphthyl) propionate (MalphaNP) residues (Rh2) is slightly better for strong donors than the complex with four Mosher acid anions (Rh1), but it is inferior for weak donors. On the other hand, the dirhodium tetracarboxylate complex with four (S)-N-phthaloyl-(S)-tert.-leucinate residues (Rh3) is generally more effective than Rh1. These results are explained by the estimated conformational behavior of the substituents within the equatorial acid residues and the anisotropy (ring-current) effect of aryl groups.

[Research agenda of interferon-gamma release assays].

Recently, new diagnostic tests for tuberculosis infection that are more specific than tuberculin skin tests have been developed and have become commercially available. These tests determine interferon-gamma production after stimulation with M. tuberculosis-specific antigens, ESAT-6 and CFP-10, and are named Interferon-Gamma Release Assays (IGRA). Currently, two IGRA formats are available. One is QuantiFERON TB-2G (QFT-2G, called Quanti FERON-TB Gold outside Japan), which uses the whole blood and has been approved as a diagnostic test for tuberculosis infection in Japan. The use of QFT-2G was recommended for contact investigations in the revised guidelines in 2006. The other format of IGRA is T-SPOT.TB (T-SPOT), which uses peripheral blood mononuclear cells. T-SPOT has not yet been approved in Japan. IGRA was developed just recently, so there are many research questions to be addressed. In 2007, Pai et al. reported a comprehensive research agenda on IGRA). We introduced a review of Pai et al. in Japanese with reference to our published reports. The references in the review of Pai et al. appear as they are, and our new references are numbered with Roman numerals.