Chiral Recognition Of Trans-4-hydroxyproline By Water-soluble Chiral Lanthanide Shift Reagent

Trans-4-hydroxy-L-proline has wide range uses and unique flavor,which can beused as biochemical reagents, spices, flavor enhancers and nutritional enhancer,mainly for juice, soft drinks, nutritional drinks etc., whereas, its enantiomer D-isomerdoes not have these effects. Such as these, many chiral enantiomers especially chiraldrugs, usually possess different biological activity and pharmacologic because ofdiscrepant optical rotation characteristics. Therefore, the identification of the chiralconfiguration and the determination of chiral enantiomer purity is essential.Nuclear Magnetic Resonance (NMR) technique is the mose common andversatile analytical method in organic chemistry, and increasingly plays an importantrole in the scientific research and production activities of pharmaceutical. NMRspectra are very useful for structural elucidation of compounds and structureidentification of moleculars. With the upgrading of spectrometer hardware and thedevelopment of software programs, NMR has been widely used in quantitativeanalysis of drugs. In recent years, it also plays an important role in study of chiralenantiomers drug.There have been developed various methods of determing the enantiomericpurity of chiral compunds using NMR technique. In this paper, a method fordetermination of enantiomeric composition of water-soluble sample was developed.The first part has a full structure elucidation of trans-4-hydroxy-proline sample.In order to identify the resonance signals of D-isomer and L-isomer effectively, chiralshift reagent was artificially added to cause magnetic field discrepancy of enantiomermolecules. The object studied in this thesis is R-4-hydroxyproline, which has verygood water soluble, thus the chiral shift reagent should be also hydrosoluble. However,this kind of hydrosoluble reagent is limited and very lack on commercial. Based on awide survey, the Sm-pdta was selected as shift reagent in this thesis, owning to its twocharacteristics. Firstly, it is hydrosoluble and is very suitable for NMR of amino acidsoluble enantiomers. Secondly, the NMR spectral line broadening caused by paramagnetic Sm³⁺is less than other lanthanide ions, make it suitable for high fieldNMR of enantiomers analysis.Sm-pdta, a water-soluble chiral shift reagent,was added to THP solution to helpisomer signal recognising. Sm³⁺ion act as a hard Lewis acid, can coordinate withoxygen and nitrogen atoms in the donor substrates, which acts as a hard Lewis base,and forms donor-acceptor complex.The magnetic field of the paramagnetic lanthanideion causes large changes in the chemical shift in the resonances of a bound donormolecule. The shifts are primarily the result of dipolar interactions of the magneticfield of lanthanide ion through space. The larger the induced shift, the better the chiralrecognition effect.The magnitude of the perturbation and the direction of the changes in chemicalshifts depend on the lanthanide ion. Therfore, we considered solution pH and molarratio of Sm-pdta and substrate as the main factors affecting the complexation oflanthanide ion and substrate. And finally a condition of pH9.6¹0.2, molar ratio0.67was determined for the best isomer signal recognition and the largest inducedchemical shift. At this condition, in L:D7:3water solution, the induced chemical shiftof H-2, H-3a, H-4is83.9Hz,46.4Hz and44.3Hz respectively in¹H NMR spectra,the induced chemical shift of carbon C-3,C-4,C-5is14.6Hz,38.3Hz, and130.3Hzrespectively in¹³C NMR spectra.The third part of the thesis studies the use of NMR in the determination ofenantiomeric excess. Firstly, the relaxation characteristics of hydrogen atoms nuclearand carbon atoms nuclear in the solution was studied by inverse-recovery method.The relaxation characteristics of atoms nuclear related with the accuracy of thequantitative results, the relaxation is more sufficient, the quantitative error is muchsmaller. The relaxation characteristics was affected by experiment parameters, thesampling period of¹H NMR and¹³C NMR is4.7s and1.3s, respectively. After then,both method are used to determine the enantiomeric excess. In the method of¹HNMR, the characteristic peaks of H-3a of L-and D-isomer as well as the H-2of D-isomer were used for quantitative analysis. Seven different ratios of L-and D-samples were analyzed, and the enantiomeric excess was compared with theory, witha correlation coefficient of0.9999, indicating that the result is reliable. In the methodof¹³C NMR, the integration areas of the C-3,C-4,C-5peaks of seven different ratiosof L-and D-samples were calculated, and the enantiomeric excess was then determined. The result shows that the C-5peak is the best one and the correlationcoefficient of seven samples is0.9994. When the L:D ration is99.5:0.5, because ofthe low concentration of D-isomer, the peaks of C-3and C-4were disturbed by lowsignal to noise ratio, the quantitative analysis has a large deviation. For other sixsamples, the peaks of C-3and C-4has a correlation coefficient of0.9993and0.9999,indicating a reliable result.Quantitative analysis by¹³C NMR is the one of the most outstanding part of thiswork. For¹³C NMR, the characteristic is the spectrum distribution is very wide, thusevery line represents a carbon with special position. There is hardly any overlap ofspectral lines and much more available peaks for quantitative analysis. Like in thethesis, the carbon peaks of C-3,C-4,C-5are both can be used for quantitativeanalysis.With the detection sensitivity improvement of probe,¹³C NMR will becomea potential method in relative quantitative determination.As to¹H NMR, when the concentration of D-isomer is very low (less than0.5%),the resonance signal of proton still can be observed and quantitatively analyzedbecause of the high sensitive of proton in¹H NMR. However, the shortcoming of¹HNMR is that the selective of characteristic peak is complexity. One should carefullystudy each peak, ensuring that each D-and L-signal has been isolated, as well as nooverlap disturb at the peak for quantitative analysis. When calculating the integrationarea of the peak, the phase needs careful adjustment to determine the starting pointand check point. If the noise per signal of the spectrum is very high, the line widthfactor should not be used. Alternatively, a liner prediction can be used to solve theproblem of high baseline or the signal below the baseline.In conclusion, the method of using chiral shift reagent for water-soluble chiralenantiomers provided in this thesis is stable and reliable. Using proper operatingconditions and¹H NMR as well as¹³C NMR, the purity of enantiomers can bedetermined accurately. This method can also be used for the analysis of other chiraldrugs.