A detailed protocol describing the SSTD NMR method is presented here to help new users apply this new method to obtain the kinetic parameters of their own systems undergoing chemical exchange.
该协议详细介绍了新的自旋饱和转移差异核磁共振协议(SSTD NMR),最近在我们的小组开发研究,是很难用传统的方法来分析相互现场的化学交换过程。顾名思义,该方法中,通过测定瞬态自旋饱和转移沿增加饱和时间(建立结合用于小分子的自旋饱和转移的方法,用饱和转移差异为蛋白 – 配体相互作用的研究采用(STD)NMR法-up曲线)在发生化学交换小的有机和有机分子。
这种方法比现有的优点是:没有必要到达交换信号的聚结;该方法可以,只要可以应用作为交换位点是分离的一个信号;没有必要测量Ť1或达到稳态饱和;速率常数VA梅毒被直接测量,并且在相同的实验中获得Ť1值,只用一组实验。
为了测试该方法,我们研究了N,N- -dimethylamides的受阻旋转,对于其可用于比较大量的数据的动态。使用SSTD得到的热力学参数非常类似于所报告的那些(自旋饱和转移技术和线形分析)。该方法可以适用于无法通过以前的方法来研究更有挑战性基板。
我们设想,在简单的实验设置,并且该方法的广泛适用性极大各种基材将使这个之间无核磁共振广泛的专业知识的有机和有机金属化学家的常用技术。
化学交换通常是指任何分子间或分子内的方法,其中一个核从一个环境移动到另一个,其中其核磁共振参数(化学位移,标量偶合,偶极耦合,松弛率)不同。有有机和有机分子( 例如,在联芳基旋转障碍,环翻转障碍和构象平衡,氮反转,配体结合,简配体交换和互变异构)化学交换的例子不胜枚举1-3化学汇率相关交换过程的屏障的热力学,因此,其研究是至关重要的,以了解这些系统的分子动力学。
核磁共振动态交换的经典标志是在NMR信号随着温度的变化的线形了戏剧性的变化。在低温下,该过程是缓慢的,两个不同的化学位移是安装前后VED。在高温下,这两个信号合并成一个信号,它被称为“聚结”。在中间温度时,信号变得非常广泛。核磁共振谱的化学交换这种敏感性使得NMR一个非常强大的方法来研究分子动力学解决方案。两种方法已被采用,主要在溶液中的动态过程的研究。线形分析,4-7和自旋饱和转移实验8-9此外,它也是值得一提的反转传输方法10,投洽会项目11直接提取速率常数,这是简单的系统交换测量相对有效的办法。虽然这些方法在大多数情况下,得到了很好的效果,它们,然而,有许多缺点。该线形态分析的主要缺点是在一些样品中达成合并所需要的高温。12,主要要考虑的问题时,CArrying出自旋饱和转移实验是:到达交换位点之间的稳态饱和转移所需的很长的饱和时间,并且需要确定的纵向弛豫时间常数,T 1,这是很困难的,如果有不同的重叠在研究的区域信号。13
作为我们在有机金属机制调查的一部分,14-16我们组正在研究铂-丙二烯络合物在溶液中的fluxional行为。这是一个复杂的任务,包括在至少三个不同的过程,其中之一是金属的丙二烯轴线之一左右的π面交换或旋转。我们遇到的正常的VT实验和已经在类似的系统之前采用线形分析技术,17-19不适合于我们的研究中,由于在我们的铂-丙二烯络合物的非常缓慢的旋转,使得的聚结温度SIG的兴趣比络合物分解的温度高良。
为了克服这一限制,我们开发和最近报道了新的NMR协议(SSTD NMR)研究相互现场的化学交换过程。20顾名思义本方法结合使用的小分子的自旋饱和转移的方法,用饱和转移差异核磁共振方法通过测量沿发生化学交换小分子日趋饱和时间(积曲线)瞬态自旋饱和转移用于蛋白质-配体相互作用的研究中,21-24。
用这种新的方法(SSTD NMR)我们已经表明,我们可以得到超过传统方法的一些额外的优点小有机和有机金属分子的分子内的化学交换的动力学参数:不需要的信号的聚结,因此,更灵活的温度范围内可以使用在研究中;信号重叠不干扰,虽然交换共振中的至少一个应隔离;没有必要测量Ť1或达到稳态饱和;速率常数的值被直接测量,并在相同的实验,获得Ť1值,只用一组实验。所述SSTD核磁共振方法的另一个显着的优点是,相对于线形分析,动力学速率常数的测定是不通过与高磁场相关联的聚结温度的增加的限制。因此,我们的方法,然后很好拨到低和高磁场。本文旨在帮助新用户使用这种新方法,以自己的系统进行化学交换,并介绍了样品制备,实验装置,数据采集,并在一个简单的有机分子的数据处理和分析的例子。
One of the more obvious advantages of this methodology is that the rate constants and the relaxation time for a given temperature can be obtained with a single set of experiments, with a robust pulse sequence (the same used for STD experiments to study protein-ligand interactions, which is typically found within the available set of experiments from the spectrometer manufacturer). This simplifies the experimental setup since there is no need to measure T1 or reach steady state saturation. Besides, it is remarkable that this method does not depend on the magnet strength, as coalescence methods. On the other hand, the main limitation is that this technique cannot be applied to chemical exchange processes too fast or too slow, which would depend on the temperature range of the NMR machine or the solvents used.
This new technique for the calculation of kinetic parameters can be applied to a great variety of substrates and its applicability has already been demonstrated with some interesting molecules.21 The kinetic parameters of the 4-N,N-dimethylamido[2.2]paracyclophane, a challenging substrate in which the signal of one of the methyl groups of interest is overlapped with other signals from the molecule, were successfully calculated using SSTD NMR. Interestingly, this methodology can be applied as long as one of the signals of study is isolated. SSTD NMR is also a useful protocol for the calculation of kinetic parameters in molecules in which the coalescence temperature is so high that the molecule decomposes before reaching it. This is the case with PtCl2(dimethylallene)(pyridine), in which the methodology was successfully applied without the need of reaching coalescence. The choice of solvents and temperatures is critical to obtain good results, since the chemical exchange rates can vary significantly with these parameters. Moreover, in addition to the criteria in a normal NMR experiment, key steps in a SSTD NMR experiment are the selectivity of the irradiation as well as the temperature control. Both factors have to be precise to guarantee the success of the experiment.
The representative results presented here are for the kinetics of intramolecular chemical exchange, but the technique can also be applied to study the kinetics of intermolecular chemical exchange and also ligand exchange, common processes in the dynamic behavior of transition metal complexes.
Finally, providing a proper modification of the equations is made,32 this method could be extended to deal with multi-site exchange and unequal populations, as it has been done in former double resonance experiments,8-9 increasing the usefulness of this technique for the study of chemical exchange processes in challenging compounds.
The authors have nothing to disclose.
Funding by the University of East Anglia, the EPSRC (EP/L012855/1) and the EU (H2020-MSCA-IF-2014-EF-ST-658172) is gratefully acknowledged (MTQ).
N,N-dimethylacetamide | Aldrich | 38840 | Acute toxicity |
Toluene-d8 | Fluorochem | D-005 | Flammable and toxic |
500MHz 7" Select Series NMR Tubes | GPE LTD | S-5-500-7 | |
TopSpin 2.1 | TopSpin program, Bruker Corp., http://www.bruker.com/products/mr/nmr/nmr-software/software/topspin/ (2015). | ||
Origin 6.0 | Origin 6.0 software, OriginLab Corp., http://originlab.com. | ||
Bruker Avance III 500 MHz fitted with 5mm broadband observed BBFOplus Z-gradient SmartProbeTM probe | Bruker Corp., http://www.bruker.com | ||
Bruker Avance I 500 MHz Inverse Triple Resonance NMR spectrometer fitted with a 5mm TXI Z-gradient probe | Bruker Corp., http://www.bruker.com | ||
Ceramic Spinner standardbore shimsystems (5 mm) | Bruker Corp., http://www.bruker.com | H00804 |