第四章 二维核磁共振谱.doc

第四章 二维核磁共振谱41二维核磁共振的概述1什么是二维谱二维核磁共振(2D NMR)方法是有Jeener 于1971年首先提出的,是一维谱衍生出来的新实验方法.引入二维后,减少了谱线的拥挤和重叠,提高了核之间相互关系的新信息.因而增加了结构信息,有利于复杂谱图的解析.特别是应用于复杂的天然产物和生物大分子的结构鉴定,2DNMR是目前适用于研究溶液中生物大分子构象的唯一技术.一维谱的信号是一个频率的函数,记为S(),共振峰分别在一条频率轴上.而二维谱是两个独立频率变量的信号函数,记为S(1,2),共振峰分布在由两个频率轴组成的平面上.2D-NMR的最大特点是将化学位移,偶合常数等参数字二维平面上展开,于是在一般一维谱中重叠在一个频率轴上的信号,被分散到两个独立的频率轴构成的二维平面上.,同时检测出共振核之间的相互作用.原则上二维谱可以用概念上不同的三种实验获得,(如图4.1),(1).频率域实验(frequency- frequency) (2).混合时域(frequency-time)实验(3). 时域(time-time)实验.它是获得二维谱的主要方法,以两个独立的时间变量进行一系列实验,得到S(t1,t2),经过两次傅立叶变换得到二维谱S(1,2).通常所指的2D-NMR均是时间域二维实验.图4.1 2D-NMR 三种获得方式2二维谱实验二维谱实验中，为确定所需的两个独立的时间变量，要用特种技术时间分割。即把整个时间按其物理意义分割成四个区间。（如图所示）图4.2 一般二维谱实验（1）预备期：预备期在时间轴上通常是一个较长的时期，使核自旋体系回复对平衡状态，在预备期末加一个或多个射频脉冲，以产生所需要的单量子或多量子相干。（2）在t1开始时由一个脉冲或几个脉冲使体系激发，此时间系控制磁化强度运动，并根据各种不同的化学环境的不同进动频率对它们的横向磁化矢量作出标识。（3）在此期间通过相干或极化的传递，建立检测条件。在此期间检测作为t2函数的各种横向矢量的FID的变化。它的初始相及幅度受到t1函数的调制。与t2轴对应的2（轴），通常是频率轴，与t1轴对应的1是什么，取决于在发展是何种过程。3二维谱的表达方式(1) 堆积图(stacked plot).堆积图的优点是直观,具有立体感.缺点是难以确定吸收峰的频率。大峰后面可能隐藏小峰，而且耗时较长。图4.3 堆积图 等高线(2)等高线（Contour plot）等高线图类似于等高线地图，这种图的优点是容易获得频率定量数据，作图快。缺点是低强度的峰可能漏画。目前化学位移相关谱广泛采用等高线。 4二维谱峰的命名（1） 交叉峰（cross peak）:出现在12处，（即非对角线上）。从峰的位置关系可以判断哪些峰之间有偶合关系，从而得到哪些核之间有偶合关系，交叉峰是二维谱中最有用的部分。（2） 对角峰（Auto peak）:位于对角线（12）上的峰，称为对角峰。对角峰在F1和F2轴的投影。图4.4 典型二维谱示意图5二维谱的分类二维谱可分为三类：1）J 分辨谱（ J resolved spectroscopy ）J 分辨谱亦称J谱或者J谱。它把化学位移和自旋偶合的作用分辨开来，包括异核和同核J谱。2）化学位移相关谱(chemical shift correlation spectroscopy)化学位移相关谱也称谱，是二维谱的核心，通常所指的二维谱就是化学位移相关谱。包括同核化学位移相关谱，异核化学位移相关谱，NOESY和化学交换。3）多量子谱（multiple quantum spectroscopy）用脉冲序列可以检测出多量子跃迁，得到多量子二维谱。42 化学位移相关谱（Correlated Spectroscopy ,COSY）二维化学位移相关谱包括：同核化学位移相关谱（Homonuclear correlation）1）通过化学键：COSY, TOCSY, 2D-INADEQUATE。2）通过空间：NOESY, ROESY。异核化学位移相关谱（Heteronuclear correlation）强调大的偶合常数：1H-13C COSY强调小的偶合常数，压制大的偶合常数：COLOC(远程1H-13C COSY)。4.2.1同核化学位移相关谱1 1H-1H-COSY 所谓的1H-1H-COSY系指同一自旋体系里质子之间的偶合相关。1H-1H-COSY可以1H-1H之间通过成键作用的相关信息，类似于一维谱同核去偶，可提供全部1H-1H之间的关联。因此1H-1H-COSY是归属谱线，推导结构及确定结构的有力工具。COSY90。的基本脉冲序列包括两个基本脉冲图4.5COSY90。的基本脉冲序列在此脉冲作用下，根据发展期t1的不同，自旋体系的各个不同的跃迁之间产生磁化传递，通过同核偶合建立同种核共振频率间连接图。此图的二个轴都是1H的在12的对角线上可以找出一维1H谱相对应谱峰信号。通过交叉峰分别作垂线及水平线与对角线相交，即可以找到相应偶合的氢核。因此从一张同核位移相关谱可找出所有偶合体系，即等于一整套双照射实验的谱图。例如(COSY 相关谱（Correlation Spectroscopy） o both axes correspond to the proton nmr spectra. o the COSY spectra indicates which H atoms are coupling with each other. 图4.7 典型 2丁烯酸乙酯COSY谱图图4。6 COSY of First look at the peak marked A in the top left corner. This peak indicates a coupling interaction between the H at 6.9 ppm and the H at 1.8 ppm. This corresponds to the coupling of the CH3 group and the adjacent H on the alkene. Similarly, the peak marked B indicates a coupling interaction between the H at 4.15 ppm and the H at 1.25 ppm. This corresponds to the coupling of the CH2 and the CH3 in the ethyl group. Notice that there are a second set of equivalent peaks, also marked A and B on the other side of the diagonal. 2 COSY 45COSY 45 的基本脉冲序列：/2-t1-/4-ACQ.即将COSY 90的第二脉冲变成45。许多天然产物的直接连接跃迁谱线落在对角线附近，导致谱线相互重叠，不易解析。COSY 45比COSY 90减少了平行跃迁磁化转移强度，即消除了对角线附近的交叉峰，使对角线附近较为清晰。图4.8 蔗糖的COSY 90 图4.9 蔗糖的COSY 45 3LR COSY(long range COSY)常规的COSY 90 脉冲序列测得的COSY谱，观察不到较小的远程偶合交叉峰。LR COSY是在COSY 90的序列基础上引入较长的延迟D2用于小J磁化转移的发展，能大大增加来自远程偶合的交叉峰的强度。其基本脉冲序列是：/2-t1-D2-ACQ(t2)图4.10 蔗糖的COSY 90和LRCOSY在COSY 90中邻位大的偶合G1和G2可以清楚看出。在LRCOSY中除了可以看出G1和G2的偶合，还可以找到G1和G3，G4的小J的偶合交叉峰。因此在LRCOSY 谱中除了可以发现大J的邻位偶合交叉峰，还存在着小J的远程偶合（3JH-H,4JH-H）,解析谱图时，应该对照COSY加以区别。4相敏COSY谱COSY谱，由于谱线信号色散分量作用，相邻的峰容易相互部分重叠，交叉峰的精细结构看不清楚，不便读出偶合常数。相敏COSY谱的相位很复杂，相位调节的质量直接影响偶合常数的检测即信号灵敏度。在COSY谱中对角线与交叉峰相位总是相差90。相敏COSY谱中，磁化转移地结果产生一对交叉峰相位相差180。以AX体系为例。其交叉峰为纯吸收线形，对角线为色散型。谱图黑色圆圈为正峰，红色为负峰。图4.12 AX体系相敏COSY谱图4.13 AMX相敏COSY谱与COSY有关的实验还有,自旋回波COSY(SECSY),双量子相干谱（DQC-COSY）,同核接力相干谱（RCT）.有兴趣的同学，可以阅读有关的书籍。3 天然丰度的双量子13C谱（INADEQUATE(13C-13C-COSY)）这是二维碳骨架直接测定法，是确定碳原子连接顺序的实验，一种双量子相干技术。是一种13C-13C化学位移相关谱。在质子去偶的13C谱中，除了13C信号外，还有比它弱200倍的13C-13C偶合卫星峰，13C-13C偶合含有丰富的分子结构和构型的信息。由于碳是组成分子骨架，它更能直接反映化学键的特征与取代情况。但是由于13C天然丰度仅仅为1.1%，出现13C-13C偶合的几率为0。01，13C-13C偶合引起的卫线通常离13C强峰只有20Hz左右，其强度又仅仅是13C强峰的1/200，这种弱峰往往出现在强13C峰的腋部，加上旋转边带，质子去偶不完全，微量杂质的影响等因素，使1JC-C测试非常困难。利用双量子跃迁的相位特性可以压住强线，突出卫线求出JC-C,并根据Jc-c确定其相邻的碳。一个碳原子最多可以有四个碳与之相连，利用双量子跃迁二维技术测量偶合碳的双量子跃迁的频率。13C-13C同核偶合构成二核体系（AX,AB）两个偶合的13C核能产生双量子跃迁，孤立的碳则不能。它只有一个双量子跃迁，其频率正比于两个偶合的13C核的化学位移之和的平均值。所以如果两个碳具有相同的双量子跃迁频率，即可以判断，它们是相邻。在INADEQUATE谱图中F1与F2分别代表双量子跃迁频率和13C的卫线，依次代表双量子和单量子跃迁频率。谱图中一个轴是13C的化学位移，一个为双量子跃迁频率，其频率正比于两个偶合的13C核的化学位移之和的平均值。因此谱图中F1=2F21的斜线两侧对称分布着两个相连的13C原子信号，表示碳偶合对的单量子平均频率与双量子频率间的关系，水平连线表明一对偶合碳具有相同的双量子跃迁频率，可以判断它们是直接相连的碳。依此类推可以找出化合物中所有13C原子连接顺序。18 - 11 - 16 - 15 - 17 - 13.异核化学位移相关谱（Heteronuclear Correlation of chemical shift）所谓异核化学位移相关谱是两个不同核的频率通过标量偶合建立起来的相关谱应用最广泛的是1H-13C COSY. 1. 1H-13C COSY.常规的1H-13C COSY是指直接相连的C-H之间的偶合相关（1JCH）。 NOESY spectrum of codeine codeine in 0.65 ml CDCl3A contour plot of the NOESY spectrum is shown below.As with all homonuclear 2D plots, the diagonal consists of intense peaks that match the normal spectrum, as do projections onto each axis. The interesting information is contained in the cross-peaks, which appear at the coordinates of 2 protons which have an NOE correlation.For small molecules, the NOE is negative. Exchange peaks have the opposite sign from NOE peaks, making them easy to identify. The water peak at 1.5 ppm exchanges with the OH at 2.9 ppm, shown here in red.The spectrum is phased with the large diagonal peaks inverted (shown in red here), so the NOE cross-peaks are positive.Expansion of the upfield region:Table of NOEs: ( indicates the more upfield of geminal CH2 protons)8 - 7, 127 - 18, 183 - 5, 105 - 11, 16, 189 - 10, 17, 1710 - 1611 - 18, 16, 14, 1818 - 13, 1816 - 14, 1713 - 14, 17, 1713 - 17, 1717 - 17In addition to confirming assignments, the NOESY spectrum allows stereospecific assignments of methylene Hs. The 3 cross-peaks indicated in red on the plot below distinguish between the 3 CH2 pairs:5 -1816 - 1718 - 13These NOEs can be viewed interactively in 3D using Chime. phase-sensitive NOESY of strychnine.TOCSYTOCSY 是总相关谱的缩写（ TOtal Correlation SpectroscopY）. 也称之为 HOHAHA (HOmonuclear HArtmann HAhn). During this pulse sequence, after the evolution period t1, the magnetization is spin-locked (for example by a series of 180 pulses). During this mixing time the magnetization exchange through scalar coupling. During this spin-lock period, the magnetization behaves as a strongly coupled spin system and evolve under the influence of a collective spin-mode. In that collective mode, coherence transfer is possible between all coupled nuclei in a spin system, (even if the are not directly coupled). One of the most popular mixing scheme in TOCSY is the MLEV-17.For small mixing period, (e.g. 20-30 msec) Relay1-COSY type of data can be observed. As the mixing period gets longer, correlation with more distant protons can be observed (e.g. mix=80-100 msec can correlate H1 to H6 in carbohydrate). The extent of correlation depends mainly on the length of the mixing period. This sequence is much more useful than the Relay-COSY as it very efficiently transfer magnetization before relaxation takes place. TOCSY （总相关谱）(Total Correlation Spectroscopy)与 COSY相似,在图中可以看到彼此偶合氢的相关。 但是TOCSY spectrum, 可以看到整个偶合体系所有的氢，不仅仅是直接偶合的氢。例如, 3-heptanone: 质子a,b,c是一个自旋体系，e,f是一个独立的自旋体系。在 COSY spectrum中, CH2 a 与 CH2 b相关. In a TOCSY spectrum, 它也与CH2s c and d相关.Codeine (3.3 mg in .65 ml CDCl3)The TOCSY spectrum of codeine is shown below. As with all homonuclear 2D plots, the diagonal (lower left corner to upper right corner) consists of intense peaks that match the normal spectrum, as do projections onto each axis. The interesting information is contained in the cross-peaks, which appear at the coordinates of 2 protons which belong to the same spin system.The cross-peaks marked with red and green circles are longer-range correlations, not observed in the COSY spectrum. The green circles mark cross-peaks to the water peak. Water in the solvent exchanges with the OH of codeine, which is coupled to H-10, which is, in turn, coupled to H-9. It takes time for the longer-range correlations to develop (referred to as magnetization transfer), so the final spectrum is dependent on delay values in the pulse sequence. In practice, several spectra may be acquired using different delay values. The intensity of each cross-peak will vary through the series of spectra - long range correlations will grow in as the delay value is increased, and shorter range couplings may disappear.Table of TOCSY peaks:( indicates the more upfield of geminal CH2 protons)8 - 73 - 5, 9, 10, 165 - 9, 10, 11, 169 - 10, 16, OH, H2O10 - 16, OH, H2O11 - 16, 18, 1818 - 16, 1816 - 1813 - 13, 17, 1713 - 17, 1717 - 17 异核化学位移相关谱（Heteronuclear Correlation of chemical shift）bCOLOCbHMQC (1-bond CH correlation) of codeine）This is a 2D experiment used to correlate, or connect, 1H and 13C peaks for directly bonded C-H pairs. The coordinates of each peak seen in the contour plot are the 1H and 13C chemical shifts. This is helpful in making assignments by comparing 1H and 13C spectra.This experiment yields the same information as the older HETCOR experiment, but is more sensitive, so can be done in less time and/or with less material. This is possible because in the HMQC experiment, the signal is detected by observing protons, rather than carbons, which is inherently more sensitive, and the relaxation time is shorter. This so-called inverse detection experiment is technically more difficult and is possible only on newer model spectrometers. Acorn NMRs new JEOL Eclipse+ 400 is equipped to perform inverse experiments, and uses Z-gradients for improved spectral quality.The time required for an HMQC depends on the amount of material, but can be done in 1/2 hour or less, compared to several hours for a HETCOR spectrum.Contour plot of the HMQC spectrum. Because it is a heteronuclear experiment, the 2 axes are different, and the plot is not symmetrical. Unlike a COSY spectrum, there are no diagonal peaks.Normal 1D 1H and 13C spectra are shown along the edges. Peaks occur at coordinates in the 2 dimensions corresponding to the chemical shifts of a carbon and its directly bonded proton(s). For example, the contour peak indicated in red shows that the 13C with peak at 91.5 ppm is bonded to the 1H with peak at 4.9 ppm.Non-equivalent methylene protons are easily identified as 2 peaks located at the same 13C position. There are 3 CH2s in the codeine HMQC spectrum. Contour plot of the HMQC spectrum. Because it is a heteronuclear experiment, the 2 axes are different, and the plot is not symmetrical. Unlike a COSY spectrum, there are no diagonal peaks.Normal 1D 1H and 13C spectra are shown along the edges. Peaks occur at coordinates in the 2 dimensions corresponding to the chemical shifts of a carbon and its directly bonded proton(s). For example, the contour peak indicated in red shows that the 13C with peak at 91.5 ppm is bonded to the 1H with peak at 4.9 ppm.Non-equivalent methylene protons are easily identified as 2 peaks located at the same 13C position. There are 3 CH2s in the codeine HMQC spectrum.1H13CAssignment6.611386.512075.713335.312854.89194.266103.856123.359113.0 & 2.320182.640162.6 & 2.446132.443142.0 & 1.83617The sample is 3.3 mg codeine in .65 ml CDCl3There are variations on this experiment, including a version in which CH2s have phase opposite of that of CH and CH3 peaks, called an HSQC-DEPT spectrum. Negative peaks are shown in red in the plot below, easily identifying the 3 CH2s in codeine.See also: detailed description of 2D processing, processing HMQC data, HMBC, comparison of HMQC and HSQC.HMBC (multiple-bond CH correlation) of codeineThis is a 2D experiment used to correlate, or connect, 1H and 13C peaks for atoms separated by multiple bonds (usually 2 or 3). The coordinates of each peak seen in the contour plot are the 1H and 13C chemical shifts. This is extremely useful for making assignments and mapping out covalent structure.The information obtained is an extension of that obtained from an HMQC spectrum, but is more complicated to analyze. Like HMQC, this is an inverse detection experiment, and is possible only on newer model spectrometers. Acorn NMRs new JEOL Eclipse+ 400 is equipped to perform inverse experiments, and uses Z-gradients for improved spectral quality.The time required for an HMBC depends on the amount of material, but is much greater than for HMQC, and can take from an hour to overnight.The contour plot shown below is of 3.3 mg codeine in .65 ml CDCl3. See also comparison to the HMBC spectrum of an 18 mg sample.Normal 1D 1H and 13C spectra are shown along the edges. Peaks occur at coordinates in the 2 dimensions corresponding to the chemical shifts of a carbon and protons separated by (usually) 2 or 3 bonds. The experiment is optimized for couplings of 8 Hz. Smaller couplings are observed, but their intensities are reduced. Compare to the spectrum obtained when the experiment is optimized for 4 Hz.The experiment is designed to suppress 1-bond correlations, but a few are observed in most spectra. In concentrated samples of conjugated systems, 4-bond correlations can be observed. There is no way to know how many bonds separate an H and C when a peak is observed, so analysis is a process of attempting to assign all observed peaks, testing for consistency and checking to be sure none of the assignments would require implausible or impossible couplings.Because of the large number of peaks observed, analysis requires several expanded plots. In this case, the spectrum has been divided into 4 sections, each of which is discussed below.The discussion below uses the numbering system shown at right. The numbers were assigned to peaks in the 1D 13C spectrum, starting downfield, moving upfield, and numbering each sequentially. This generates a unique identifier for each Carbon, even before knowing any assignments. In aromatic rings, the most common correlations seen in HMBC spectra are 3-bond correlations because they are typically 7-8 Hz, which is the value for which the experiment is optimized. The coupling constant is affected by substituents, so 2-bond correlations are also sometimes observed.The red lines in the plot above show correlations from aromatic proton H-8 to aromatic carbons C-1 and C-6 (both are 3-bond couplings) and a weak correlation to C-2, a 2-bond coupling.The other aromatic proton, H-7, has correlations to C-2 and C-4, both of which are 3-bond couplings.The green lines in the plot above show correlations from proton H-9 to carbons C-1, C-3 and C-4 (all are 3-bound couplings). With the poor digital resolution of the spectrum in the carbon dimension (512 data points spread over 17kHz), the peaks for C-3 and C-4 run together because they are barely resolved. The peaks indicated in red above are due to 1-bond coupling in CHCl3 solvent. Note that the pair of peaks dont line up with any H peaks, but