(1)H NMR based profiling of spent culture media cannot predict success of implantation for day 3 human embryos.

BACKGROUND: Identification of a non-invasive technique to assess embryo implantation potential in assisted reproduction would greatly increase success rates and lead more efficiently to single embryo transfer. Early studies suggested metabonomic analysis of spent culture media could improve embryo selection. The goal of this study is to assess if embryo implantation can be predicted based on proton nuclear magnetic resonance ((1)H NMR) profiles of spent embryo culture media from patients undergoing transfer of multiple embryos on cycle day 3. METHOD: We conducted a retrospective study in an academic assisted reproduction technology (ART) program and analyzed the data in a university research center. Two hundred twenty-eight spent culture media samples originating from 108 patients were individually analyzed. Specifically, five distinct sets (1 to 5) of different types of spent media samples (volume ~14 µL) from embryos that resulted in clinical pregnancy (positive heart rate at 6 weeks gestation) (n (1) = 29; n (2) = 19; n (3) = 9; n (4) = 12; n (5) = 33; n (total) = 102) and from embryos that did not implant (n (1) = 28; n (2) = 29; n (3) = 18; n (4) = 15; n (5) = 36; n (total) = 126) were collected on day 3 of embryo growth. The media samples were profiled using (1)H NMR spectroscopy, and the NMR profiles of sets 1 to 5 were subject to standard uni- and multi-variate data analyses in order to evaluate potential correlation of profiles with implantation success. RESULTS: For set 1 of the media samples, a borderline class separation of NMR profiles was obtained by use of principal component analysis (PCA) and logistic regression. This tentative class separation could not be repeated and validated in any of the other media sets 2 to 5. CONCLUSIONS: Despite the rigorous technical approach, (1)H NMR based profiling of spent culture media cannot predict success of implantation for day 3 human embryos.

Selective double quantum resolved correlation experiment for the complete separation of entire proton NMR spectra of enantiomers.

The present study reports a two dimensional NMR experiment which separates single quantum spectra of enantiomers from that of a racemic mixture. This is a blend of selective double quantum refocusing, for resolving couplings and chemical shift interactions along two dimensions followed by correlation of the selectively excited protons to the entire coupled spin network. The concept is solely based on the presence of distinct intra methyl dipolar couplings of different enantiomers when dissolved in chiral orienting media. The analysis of single enantiomer spectrum obtained from respective F(2) cross sections yield all the spectral information.

Synthesis and studies of thiacorroles.

The first examples of stable 22-thiacorroles containing a direct pyrrole-pyrrole bond were synthesized using thiophene monocarbinols as key precursors. Condensation of 1 equiv of thiophene monocarbinol with 1 equiv of substituted aldehyde and 1.5 equiv of pyrrole under refluxing propionic acid conditions followed by alumina column chromatographic purification yielded the desired 22-thiacorroles. Although the corrole formation had been observed with variety of substituted aldehydes, the stable thiacorroles were isolated in 2-3% yields only with 4- and 3-nitrobenzaldehydes. The stable thiacorroles were obtained only under harsh propionic acid conditions and any other mild reaction conditions did not yield 22-thiacorroles. The structure of thiacorroles were unambiguously established using mass, and 1D and 2D NMR spectroscopy. The NMR study indicated diminished ring current and distortion of 22-thiacorroles. DFT studies also supported the distortion of thiacorroles. The absorption spectra showed reduction in number of absorption bands and fluorescence study indicated that 22-thiacorroles are weakly fluorescent. The electrochemical studies indicated that 22-thiacorroles undergo easier reductions compared to thiaporphyrins.

Selective homonuclear decoupling in 1H NMR: application to visualization of enantiomers in chiral aligning medium and simplified analyses of spectra in isotropic solutions.

The proton NMR spectral complexity arising due to severe overlap of peaks hampers their analyses in diverse situations, even by the application of two-dimensional experiments. The selective or complete removal of the couplings and retention of only the chemical shift interactions in indirect dimension aids in the simplification of the spectrum to a large extent with little investment of the instrument time. The present study provides precise enantiodiscrimination employing more anisotropic NMR parameters in the chiral liquid crystalline medium and differentiates the overlapped peaks of many organic molecules and peptides dissolved in isotropic solvents.

Application of z-COSY experiment and its variant for accurate chiral discrimination by (1)H NMR.

We report the application of z-COSY experiment and a band selected version of it by employing a selective 90 degrees pulse entitled BASE-z-COSY for precise chiral discrimination, quantification of enantiomeric excess and the analyses of the (1)H NMR spectra of chiral molecules aligned in the chiral liquid crystalline solvent poly-gamma-benzyl-l-glutamate (PBLG). We have demonstrated their applicability for obtaining very high resolution in the (1)H NMR spectra of small organic molecules. It is well known that the commonly employed z-COSY experiment disentangles the spectral complexity, provides pure phase spectra with high resolution, aids in the complete spectral analyses, in addition to yielding information on relative signs of the couplings. The BASE-z-COSY experiment possesses all these properties, permits the measure of enantiomeric excess, in addition to large saving of instrument time.

Simplifying the complex 1H NMR spectra of fluorine-substituted benzamides by spin system filtering and spin-state selection: multiple-quantum-single-quantum correlation.

The proton NMR spectra of fluorine-substituted benzamides are very complex (Figure 1) due to severe overlap of (1)H resonances from the two aromatic rings, in addition to several short and long-range scalar couplings experienced by each proton. With no detectable scalar couplings between the inter-ring spins, the (1)H NMR spectra can be construed as an overlap of spectra from two independent phenyl rings. In the present study we demonstrate that it is possible to separate the individual spectrum for each aromatic ring by spin system filtering employing the multiple-quantum-single-quantum correlation methodology. Furthermore, the two spin states of fluorine are utilized to simplify the spectrum corresponding to each phenyl ring by the spin-state selection. The demonstrated technique reduces spectral complexity by a factor of 4, in addition to permitting the determination of long-range couplings of less than 0.2 Hz and the relative signs of heteronuclear couplings. The technique also aids the judicious choice of the spin-selective double-quantum-single-quantum J-resolved experiment to determine the long-range homonuclear couplings of smaller magnitudes.

Band selective small flip angle COSY: a simple experiment for the analyses of 1H NMR spectra of small chiral molecules.

The NMR spectroscopic discrimination of enantiomers in the chiral liquid crystalline solvent is more often carried out using (2)H detection in its natural abundance. The employment of (1)H detection for such a purpose is severely hampered due to significant loss of resolution in addition to indistinguishable overlap of the spectra from the two enantiomers. This study demonstrates that the band selected small flip angle homonuclear correlation experiment is a simple and robust technique that provides unambiguous discrimination, very high spectral resolution, reduced multiplicity of transitions, relative signs of the couplings and enormous saving of instrument time.

Separation and complete analyses of the overlapped and unresolved 1H NMR spectra of enantiomers by spin selected correlation experiments.

NMR spectroscopic discrimination of optical enantiomers is most often carried out using (2)H and (13)C spectra of chiral molecules aligned in a chiral liquid crystalline solvent. The use of proton NMR for such a purpose is severely hindered due to the spectral complexity and the significant loss of resolution arising from numerous short- and long-distance couplings and the indistinguishable overlap of spectra from both R and S enantiomers. The determination of all the spectral parameters by the analyses of such intricate NMR spectra poses challenges, such as, unraveling of the resonances for each enantiomer, spectral resolution, and simplification of the multiplet pattern. The present study exploits the spin state selection achieved by the two-dimensional (1)H NMR correlation of selectively excited isolated coupled spins (Soft-COSY) of the molecules to overcome these problems. The experiment provides the relative signs and magnitudes of all of the proton-proton couplings, which are otherwise not possible to determine from the broad and featureless one-dimensional (1)H spectra. The utilization of the method for quantification of enantiomeric excess has been demonstrated. The studies on different chiral molecules, each having a chiral center, whose spectral complexity increases with the increasing number of interacting spins, and the advantages and limitations of the method over SERF and DQ-SERF experiments have been reported in this work.

Binuclear spin state selective detection of 1H single quantum transitions using triple quantum coherence: a novel method for enantiomeric discrimination.

In the present work a novel methodology is developed for the unambiguous discrimination of enantiomers aligned in chiral liquid crystalline media and the simultaneous determination of 1H-1H and 13C-1H couplings in a single experiment. An INEPT transfer and back transfer of magnetization to protons retain the 13C edited 1H magnetization which is utilized to generate spin selective homonuclear triple quantum coherence of dipolar coupled methyl protons. Spin selective correlation of triple quantum to single quantum coherence results in spin state selective detection by 13C spin and the remaining passive protons. The difference between the successive transitions in the triple quantum dimension pertains to sum of the passive couplings and results in enhanced resolution by a factor of three. This results in unambiguous chiral visualization. The masked 13C satellite transitions in the single quantum spectrum are extracted for chiral discrimination. The technique retains all the passive homo- and heteronuclear couplings in the triple quantum dimension by the application of non-selective refocusing pulse on 1H as well as on 13C spins. This, however, refocuses the chemical shift evolution in the triple quantum dimension, and also overcomes the problem of field inhomogeneity. The method enables the determination of spectral information which is otherwise not possible to derive from the broad and featureless proton spectra. The elegant experimental technique has been demonstrated on different chiral molecules.

Spin state selective coherence transfer: a method for discrimination and complete analyses of the overlapped and unresolved 1H NMR spectra of enantiomers.

In general, the proton NMR spectra of chiral molecules aligned in the chiral liquid crystalline media are broad and featureless. The analyses of such intricate NMR spectra and their routine use for spectral discrimination of R and S optical enantiomers are hindered. A method is developed in the present study which involves spin state selective two dimensional correlation of higher quantum coherence to its single quantum coherence of a chemically isolated group of coupled protons. This enables the spin state selective detection of proton single quantum transitions based on the spin states of the passive nuclei. The technique provides the relative signs and magnitudes of the couplings by overcoming the problems of enantiomer discrimination, spectral complexity and poor resolution, permitting the complete analyses of the otherwise broad and featureless spectra. A non-selective 180 degrees pulse in the middle of MQ dimension retains all the remote passive couplings. This accompanied by spin selective MQ-SQ conversion leads to spin state selective coherence transfer. The removal of field inhomogeneity contributes to dramatically enhanced resolution. The difference in the cumulative additive values of chemical shift anisotropies and the passive couplings, between the enantiomers, achieved by detecting Nth quantum coherence of N magnetically equivalent spins provides enhanced separation of enantiomer peaks. The developed methodology has been demonstrated on four different chiral molecules with varied number of interacting spins, each having a chiral centre.

Chemical shift anisotropy edited complete unraveling of overlapped 1H NMR spectra of enantiomers: application to small chiral molecules.

The differential values of NMR spectral parameters like chemical shift anisotropies, dipolar couplings and quadrupolar couplings of enantiomers in chiral liquid crystalline media are employed not only for their visualization but also for their quantification. Large differences in chemical shift anisotropies and the quadrupolar couplings between the enantiomers enable the use of 13C and extensive 2H NMR detection for such a purpose. In spite of high magnetic moment, high sensitivity and abundant presence of protons in all the chiral molecules, 1H detection is not routinely employed due to severe overlap of unresolved transitions arising from short and long distance couplings. Furthermore, the doubling of the spectra from two enantiomers and their indistinguishable overlap due to negligible difference in chemical shift anisotropies hampers their discrimination. The present study demonstrates the use of proton chemical shift anisotropy as an exclusive parameter for such a discrimination. The method employs the non-selective excitation of homonuclear Nth quantum coherence of N coupled protons. The simultaneous flipping of all the coupled spins results in a single transition in the multiple quantum dimension at the cumulative sum of their anisotropic chemical shifts for each enantiomer, with the measurable difference between them, resulting in their complete unraveling.

Enantiomeric discrimination by double quantum excited selective refocusing (DQ-SERF) experiment.

The differences in chemical shift anisotropies, dipolar couplings, and quadrupolar couplings of two enantiomers in the chiral liquid crystalline media are employed to visualize enantiomers. In spite of the fact that proton has high magnetic moment and is abundantly present in all the chiral molecules, 1H NMR is not exploited to its full potential because of severe overlap of unresolved transitions arising from long- and short-distance couplings. Furthermore, the two spectra from R and S enantiomers result in doubling of the number of observable transitions. The present study demonstrates the application of the selectively excited homonuclear double quantum (DQ) coherence correlated to its single quantum coherence of an isolated methyl group in a chiral molecule. The DQ dimension retains only the passive couplings within the protons of the methyl group while the long-distance passive couplings are refocused, removing the overlap of central transitions, and each enantiomer displays a doublet instead of a triplet unlike in regular selective refocusing experiment. The doublet separation being different for each enantiomer results in their discrimination. The cross section taken along the single quantum dimension pertaining to each transition in the DQ dimension provides the one-dimensional spectra for each individual enantiomer with the complete removal of the overlapped transitions from the other enantiomer. The experiment is robust, the pulse sequence is easy to implement, and the methodology has been demonstrated on different chiral molecules.