水溶性镧系位移试剂对反式-4-羟基脯氨酸的手性识别
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摘要
反式-4-羟基脯氨酸的L型异构体具有广泛的用途和独特的风味，可作生化试剂、香原料、增味剂、营养强化剂等，主要用于果汁、清凉饮料、营养饮料等；而其对映体D型异构体没有这些功效。诸如此类的例子非常多。一对手性对映体，特别是很多手性药物，常常因旋光性质的不同而引起生物活性、药效上的差异。因此，识别药物的手性构型，测定手性药物的手性对映体纯度的工作至关重要。
核磁共振(NMR)技术是化合物结构解析的一种常用手段，在药学领域科研和生产活动中发挥着越来越重要的作用。核磁谱图可为化合物的结构解析提供丰富有用的信息，为分子的结构鉴定提供重要的依据。随着谱仪硬件的升级和软件程序的开发应用，核磁共振技术越来越多地应用于药物的定量分析。近几年来，核磁共振技术在药物手性对映体的研究中也扮演着重要的角色。本工作在实验室已有条件的基础上，研究手性位移试剂对反式-4羟基脯氨酸对映体的手性识别。
论文第一部分详细解析了反式-4-羟基脯氨酸的核磁谱图特征。为了在谱图上有效识别D型和L型的共振信号，采用加入手性位移试剂的方法，人为地造成对映体分子磁环境差异。本论文的研究对象反式-4-羟基脯氨酸具有很好的水溶性，因此加入的手性位移试剂也应当能溶解于水。然而目前文献报道的水溶性位移试剂种类有限，已商品化的试剂更少。经调研，本论文选取Sm-pdta作为位移试剂，它具备了两个方面的特点。一是水溶性好，适合氨基酸类水溶性对映体的核磁分析。二是由Sm~(3+)顺磁金属离子造成的核磁谱线增宽较其他镧系金属离子小，适合在高场核磁共振谱仪上进行对映体分析。
Sm~(3+)金属离子通过与底物反式-4-羟基脯氨酸分子中的含有孤对电子的氧原子、氮原子发生配位作用，造成底物分子的原子核磁环境受Sm~(3+)金属离子的干扰而发生化学位移偏移(Δδ)。底物分子中手性位点上各取代基团距中心离子的距离、空间取向存在差异，受螯合的金属离子偶极磁场诱导产生的化学位移偏移也不同。诱导化学位移越大，对映体手性识别效果越好。
为了使诱导化学位移最大，我们分别分析了溶液酸碱度，位移试剂与底物摩尔比两个因素对手性识别效果的影响。实验结果表明，溶液pH影响Sm~(3+)金属离子的螯合能力，pH过低，底物分子内羧基离子化不完全，与Sm~(3+)金属离子螯合作用弱，诱导化学位移差值偏小；pH过高，Sm~(3+)金属离子与强配位离子OH~-发生不必要的络合，同样影响了Sm~(3+)金属离子与底物分子的配位作用。因此，以对映体化学位移差值ΔΔδ为评价标准，最终选择最佳手性识别pH范围为9.6~10.2。研究位移试剂与底物摩尔比，可以发现，摩尔比并不是越大越好。摩尔比太大，对映体化学位移差值（ΔΔδ）大，但谱峰位移，造成相邻谱峰重叠。摩尔比太小，则对映体信号区分不明显。因此，综合评价化学位移差值和谱峰重叠两个因素，我们最终确定了位移试剂与底物的摩尔比为0.67。在pH9.6~10.2，摩尔比为0.67的条件下，L/D=7:3时，氢谱中对映体4位质子的ΔΔδ为44.3Hz，2位质子的ΔΔδ为83.9Hz，3a位质子的ΔΔδ为46.4Hz。碳谱中，3位碳原子的ΔΔδ为14.6Hz,4位碳原子的ΔΔδ为38.3Hz,5位碳原子的ΔΔδ为130.3Hz。
论文的第三部分考察了核磁共振在对映体过量值测定中的应用。首先，实验采用反转恢复法考察了溶液体系中氢原子核、碳原子核的弛豫特征。原子核的弛豫特征关系到定量结果的准确性，弛豫越充分，定量误差越小。原子核的弛豫性质反映在实验参数的设定上，对于氢谱，设定采样周期为4.7s；对于碳谱，设定采样周期为1.3s。然后，实验分别利用~1H NMR和~(13)C NMR方法测定对映体过量值。在~1H NMR方法中，采用L型和D型对映体的3a位质子信号以及D型异构体的2位质子信号为定量特征峰，计算了7个不同L、D配比的样品的对映体过量值，与理论ee值作线性分析，得到线性相关系数为0.9999，这说明了定量结果稳定可靠。在~(13)C NMR方法中，积分计算了7个不同L、D配比的样品中L型和D型的3位、4位、5位碳谱谱峰面积，然后根据积分面积计算对映体过量值，实验结果表明，5位碳的定量效果最佳，7个数据点线性关系良好，相关系数为0.9994；3位碳和4位碳在L:D比值为99.5:0.5时，受碳原子检测灵敏度的限制，低浓度D构型谱峰信噪比差，定量结果偏差较大。因此，将99.5:0.5这一组的定量数据除外，对余下的6个数据作线性分析，3位碳、4位碳的相关系数分别为0.9999和0.9993，定量数据可靠。
利用碳谱相对定量法测定手性对映体纯度，是目前核磁共振对映体分析中比较少用的方法，也是本论文的重点工作之一。碳谱定量最显著的优点是信号分布范围宽，每一条碳谱谱线代表一个位置碳原子，很少出现碳信号重叠，可选的定量峰较多。正如本实验中所证明的一样，3位，4位，5位碳信号均可以作为定量峰。随着探头检测灵敏度不断提高，~(13)C NMR法将成为一种潜在的相对定量分析方法。
对于氢谱定量，由于氢核检测灵敏度高，是目前大多数文献采用的方法。在D型异构体浓度较低（0.5%）时，在氢谱中仍然能够观测到质子的共振信号，且定量结果可靠。然而氢谱定量的一个缺点是氢谱谱峰重叠不可避免，这导致特征峰选择困难，需要仔细分析各个谱峰，保证定量峰的D型、L型信号完全分离。然而碳谱定量受到检测灵敏度的限制。
综上，本论文所建立的手性位移试剂对水溶性手性对映体的识别条件和方法稳定可靠，采用~1H NMR法和~(13)C NMR法测定对映体纯度结果准确可靠，可为其他同类型的手性药物分析提供方法学参考。
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 Sm~(3+)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. Sm~(3+)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 pH9.6~10.2, 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 in~1H NMR spectra,the induced chemical shift of carbon C-3,C-4,C-5is14.6Hz,38.3Hz, and130.3Hzrespectively in~(13)C 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 of~1H NMR and~(13)C NMR is4.7s and1.3s, respectively. After then,both method are used to determine the enantiomeric excess. In the method of~1HNMR, 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 methodof~(13)C 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 by~(13)C NMR is the one of the most outstanding part of thiswork. For~(13)C 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，~(13)C NMR will becomea potential method in relative quantitative determination.
As to~1H 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 in~1H NMR. However, the shortcoming of~1HNMR 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 and~1H NMR as well as~(13)C NMR, the purity of enantiomers can bedetermined accurately. This method can also be used for the analysis of other chiraldrugs.
核磁共振(NMR)技术是化合物结构解析的一种常用手段，在药学领域科研和生产活动中发挥着越来越重要的作用。核磁谱图可为化合物的结构解析提供丰富有用的信息，为分子的结构鉴定提供重要的依据。随着谱仪硬件的升级和软件程序的开发应用，核磁共振技术越来越多地应用于药物的定量分析。近几年来，核磁共振技术在药物手性对映体的研究中也扮演着重要的角色。本工作在实验室已有条件的基础上，研究手性位移试剂对反式-4羟基脯氨酸对映体的手性识别。
论文第一部分详细解析了反式-4-羟基脯氨酸的核磁谱图特征。为了在谱图上有效识别D型和L型的共振信号，采用加入手性位移试剂的方法，人为地造成对映体分子磁环境差异。本论文的研究对象反式-4-羟基脯氨酸具有很好的水溶性，因此加入的手性位移试剂也应当能溶解于水。然而目前文献报道的水溶性位移试剂种类有限，已商品化的试剂更少。经调研，本论文选取Sm-pdta作为位移试剂，它具备了两个方面的特点。一是水溶性好，适合氨基酸类水溶性对映体的核磁分析。二是由Sm~(3+)顺磁金属离子造成的核磁谱线增宽较其他镧系金属离子小，适合在高场核磁共振谱仪上进行对映体分析。
Sm~(3+)金属离子通过与底物反式-4-羟基脯氨酸分子中的含有孤对电子的氧原子、氮原子发生配位作用，造成底物分子的原子核磁环境受Sm~(3+)金属离子的干扰而发生化学位移偏移(Δδ)。底物分子中手性位点上各取代基团距中心离子的距离、空间取向存在差异，受螯合的金属离子偶极磁场诱导产生的化学位移偏移也不同。诱导化学位移越大，对映体手性识别效果越好。
为了使诱导化学位移最大，我们分别分析了溶液酸碱度，位移试剂与底物摩尔比两个因素对手性识别效果的影响。实验结果表明，溶液pH影响Sm~(3+)金属离子的螯合能力，pH过低，底物分子内羧基离子化不完全，与Sm~(3+)金属离子螯合作用弱，诱导化学位移差值偏小；pH过高，Sm~(3+)金属离子与强配位离子OH~-发生不必要的络合，同样影响了Sm~(3+)金属离子与底物分子的配位作用。因此，以对映体化学位移差值ΔΔδ为评价标准，最终选择最佳手性识别pH范围为9.6~10.2。研究位移试剂与底物摩尔比，可以发现，摩尔比并不是越大越好。摩尔比太大，对映体化学位移差值（ΔΔδ）大，但谱峰位移，造成相邻谱峰重叠。摩尔比太小，则对映体信号区分不明显。因此，综合评价化学位移差值和谱峰重叠两个因素，我们最终确定了位移试剂与底物的摩尔比为0.67。在pH9.6~10.2，摩尔比为0.67的条件下，L/D=7:3时，氢谱中对映体4位质子的ΔΔδ为44.3Hz，2位质子的ΔΔδ为83.9Hz，3a位质子的ΔΔδ为46.4Hz。碳谱中，3位碳原子的ΔΔδ为14.6Hz,4位碳原子的ΔΔδ为38.3Hz,5位碳原子的ΔΔδ为130.3Hz。
论文的第三部分考察了核磁共振在对映体过量值测定中的应用。首先，实验采用反转恢复法考察了溶液体系中氢原子核、碳原子核的弛豫特征。原子核的弛豫特征关系到定量结果的准确性，弛豫越充分，定量误差越小。原子核的弛豫性质反映在实验参数的设定上，对于氢谱，设定采样周期为4.7s；对于碳谱，设定采样周期为1.3s。然后，实验分别利用~1H NMR和~(13)C NMR方法测定对映体过量值。在~1H NMR方法中，采用L型和D型对映体的3a位质子信号以及D型异构体的2位质子信号为定量特征峰，计算了7个不同L、D配比的样品的对映体过量值，与理论ee值作线性分析，得到线性相关系数为0.9999，这说明了定量结果稳定可靠。在~(13)C NMR方法中，积分计算了7个不同L、D配比的样品中L型和D型的3位、4位、5位碳谱谱峰面积，然后根据积分面积计算对映体过量值，实验结果表明，5位碳的定量效果最佳，7个数据点线性关系良好，相关系数为0.9994；3位碳和4位碳在L:D比值为99.5:0.5时，受碳原子检测灵敏度的限制，低浓度D构型谱峰信噪比差，定量结果偏差较大。因此，将99.5:0.5这一组的定量数据除外，对余下的6个数据作线性分析，3位碳、4位碳的相关系数分别为0.9999和0.9993，定量数据可靠。
利用碳谱相对定量法测定手性对映体纯度，是目前核磁共振对映体分析中比较少用的方法，也是本论文的重点工作之一。碳谱定量最显著的优点是信号分布范围宽，每一条碳谱谱线代表一个位置碳原子，很少出现碳信号重叠，可选的定量峰较多。正如本实验中所证明的一样，3位，4位，5位碳信号均可以作为定量峰。随着探头检测灵敏度不断提高，~(13)C NMR法将成为一种潜在的相对定量分析方法。
对于氢谱定量，由于氢核检测灵敏度高，是目前大多数文献采用的方法。在D型异构体浓度较低（0.5%）时，在氢谱中仍然能够观测到质子的共振信号，且定量结果可靠。然而氢谱定量的一个缺点是氢谱谱峰重叠不可避免，这导致特征峰选择困难，需要仔细分析各个谱峰，保证定量峰的D型、L型信号完全分离。然而碳谱定量受到检测灵敏度的限制。
综上，本论文所建立的手性位移试剂对水溶性手性对映体的识别条件和方法稳定可靠，采用~1H NMR法和~(13)C NMR法测定对映体纯度结果准确可靠，可为其他同类型的手性药物分析提供方法学参考。
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 Sm~(3+)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. Sm~(3+)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 pH9.6~10.2, 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 in~1H NMR spectra,the induced chemical shift of carbon C-3,C-4,C-5is14.6Hz,38.3Hz, and130.3Hzrespectively in~(13)C 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 of~1H NMR and~(13)C NMR is4.7s and1.3s, respectively. After then,both method are used to determine the enantiomeric excess. In the method of~1HNMR, 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 methodof~(13)C 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 by~(13)C NMR is the one of the most outstanding part of thiswork. For~(13)C 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，~(13)C NMR will becomea potential method in relative quantitative determination.
As to~1H 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 in~1H NMR. However, the shortcoming of~1HNMR 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 and~1H NMR as well as~(13)C NMR, the purity of enantiomers can bedetermined accurately. This method can also be used for the analysis of other chiraldrugs.
引文
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