X-ray Crystallography Technique Analysis

X-ray Crystallography Technique Analysis1 Limitations of x-ray crystallographyFrom the foremost crystalline anatomical structure determination of table coarseness in 1914 whose structure elucidation proved the existence of ionic manifolds (6), single crystal x-ray diffraction (SC-XRD) has been widening our view of the hidden world of molecular(a) structures. Today, SC-XRD continues to be the only structural analysis method that offers direct structural information at the atomic level. As such, this technique has been vital for reliably solving some(prenominal) structures of small molecules such as neurotransmitters, antibiotics and industrial catalysts.SC-XRD utilises the ability of crystalline atoms to scatter or diffract a beam of incident x-ray into a serial publication of amplified and spatially constrained beams (3). The angles and intensities of these beams piece of tail be measured and computationally processed by a crystallographer to make grow a 3-dimensional image of the density of electrons in the crystal. away from the expertise required to process the reflection data produced, the fundamental requirement of crystals for this technique acts as major limitation, since single molecules scatter the incident x-ray to produce a weak, continuous beam that provides little useful information for analysis. While technological advances in recent decades including highly intense x-ray beams produced by synchrotrons and the teaching of more powerful algorithms for molecular structure imaging have allowed the size of the crystal required to be increasingly smaller, the need for a crystal has keep mum not been eliminated. This poses a great issue as many stub compounds atomic number 18 very difficult to crystallise, thus requiring experienced specialists while others will barely not crystallise at all. In 2013, a new protocol, later coined the crystalline sponge method (CSM), was reported that attempted to bypasses this intrinsic limitation of the ta rget molecule needing to be crystalline (1).2 The journey of the crystalline sponge methodExpectationsFujita and his team described the new method that promised to speed up SC-XRD drastically by eliminating the crystallization timber of the target molecule. This was done using porous metal organic frameworks (MOFs) that act as crystalline sponges. Due to the high molecular recognition capability of their pores, these sponges can absorb target molecules from the sample tooth root into their pores. In their study, Fujita and his team used dickens MOFs synthesised from tris(4-pyridyl)-1,3,5-triazine (TTP, 1) and the appropriate metal salt as their crystalline sponges (Co(NCS)2)3(TTP)4x( effect)n (2) and (ZnI2)3(TTP)2x(solvent)n (3). In both abstrusees, the void spaces showed strong natural covering properties for incoming customer molecules making them ideal crystalline sponges. The as-synthesized complexes2 and 3 contained solvents in the void. By soaking the crystals of 2 and 3 in a guest solution, guest molecules loathly penetrate these wet cavities by guest exchange, and are concentrated at the molecular-recognition pockets surrounded by TTP. A characteristic of the strong host-guest interaction in the crystals of 2 and 3 lies in panel ligand1, which attracts various guests onto its electron-deficient -plane. The slow guest exchange allows for the process to remain under thermodynamic control, rendering the geometry of the included guests to be regularly ordered and well equilibrated, thus making it workable to analyse the accommodated guests by crystallography since the molecular structure of the absorbed guest will be displayed, along with the host framework.Since theoretically, only one crystal is needed to suffice the experiment, Fujitas team found that even trace sample amounts of the microgram-nanogram scale can be analysed in this protocol. When the team used only 80ng of guaiazulene guest sample with a crystal of 3 (80-80-80m3), they were sur prised to see the guestwas still clearly observed. Considering that the experiment was carried forth using a laboratory X-ray machine, it seemed promising to accomplish crystallography with synchrotron X-ray experiments even on a mass of In order to assess the scope of the method, the team carried out blind crystallographic analysis of six appropriate samples (Fig) with only 5g of each sample. In conjunction with mass spectroscopic data, all six structures were decent assigned, with three of the structures solved solely from the crystallographic data. Additionally, the protocol was successfully used to determine the out-and-out(a) stereochemistry of santonin 4, an anthelminthic drug bearing four chiral centres. Unlike common absolute structure determinations, this was achieved without the chemical substance introduction of heavy atoms on the guest skeleton since the host framework contains heavy atoms (Zn and I) that show enhanced anomalous scattering effects. (ExpandThe close t o impressive result of the teams protocol however was determining the absolute structure of miyakosyneA 5, a scarce natural marine product recently obscure from a marine sponge Petrosia sp. The structure contains three chiral centres on its main alkyl chain, two of which, C3 and C26, had been previously determined to be 3R and 26R respectively. However, since the difference between the two long alkyl groups on C14 is only one methylene unit, determining the absolute configuration at C14 was ineffective by conventional spectroscopic and chemical methods. As the amount of miyakosyneA was very limited, provision a single crystal for X-ray crystallography would propose a huge challenge. The team were able throw their method to the full characterization of miyakosyneA to determine the absolute configuration at C14 and reported success. For its appraisers, it was this result that made this new protocol transformational (4) and understandably it lead to a lot of excitement in the fiel d.1.3 The FallThe sign lustre of the protocol was dulled as Fujita and his team published a correction on the sign report later that year (1b). Previously unnoticed ambiguities in the crystallographic data, alongside further investigation of by the team found the initial appellative of stereochemistry at C14 of 5 to have been incorrect. Synthetic studies by the team determined the methyls stereochemistry was opposite to the original assignment reported. Poor data quality was concluded to be the cause of this errors.Additionally, more problems were met as other research groups tried to use the technique in their own labs. Although success with the technique was achieved for simple molecules, in the first few months, other groups found little success with any interesting structures, particularly large molecules or molecules containing alkaline chemical groups (8b). Fujitas team were able to economic aid other industrial and academic groups to master the technique in one to two wee ks. Additionally, more of the issues in reproducibility were improved by the release of a more detailed report of the method (1c) that described the sponge synthesis, pore-solvent exchange and selection requirements for high quality single crystals for crystallography. However, this did not address the issue of miserable data quality that led to the misassignment of 5. Since poor data quality can be attributed to all steps of the CSM, including cystal synthesis, solvent exchange, guest-soaking, data collection and crystallographic refinement of the host-guest complex molecules in order to move the CSM from the fascinating idea phase into becoming the transformational and reliable new technology it was envisioned to be, much work was required to optimise all these steps.3. (ZnI2)3(TTP)2x(solvent)n The most successful sponge to date3.1 Andvantages of (ZnI2)3(1)2x(solvent)nIn their initial paper, Fujita and his team reported using sponges 2 and 3. With further investigation, in the c ase of complex 2, it was observed that guest molecules absorbed in the sponge were prone to static disorder as they tend to lay on the concord elements of the cubic lattice (Fm3m). Additionally, complex 2 was shown to undergo unfavourable transformations when removed from solution (8c). This destabilising transformation, accompanied by a colour change from orange to green, resulted in a semiamorphous solid with a significantly altered coordination environment at the metal centre. As such, the less symmetric (C2/c) complex 3 has been employed as the primary host complex for the crystalline sponge. The versatility of 3 as a crystal sponge stems from several advantages in host-guest complexation in the pores. Firstly, the size of the pores is ideal for cooperative organic molecules of common sizes, while the hydrophobic nature of the pore cavities provides favourable binding of common organic molecules. Also, ligand 1 in the complex offers flat and electron-deficient binding site, ap propriate for stacking with resonant compounds and for CH- interactions even with aliphatic compounds (9). Since the I atoms in the ZnI2 are good hydrogen-bond acceptors and the pyridyl protons of the ligand 1 are good hydrogen-bond donors, they provide efficient binding sites with hydrogen-bonding. Finally, the framework of sponge 3 is reatively flexible with the size of the guest not strictly limited to the pore size of the complex. Molecules larger than the portal are oftentimes accommodated by expanding the pore size. (1.3)3.2.1 Synthesis of (ZnI2)3(1)2x(solvent)n and solvent exchange by Fujita method and updated Clardy methodIn their investigations, Fujita and co-workers prepared 3 by layering a solution of zinc iodide in methanol onto a denser solution of TTP (1) in nitrobenzene. The solution is allowed to stand for 7 days, over which crystals form at the boundary of the two solvents as they diffuse before dropping to the bottom of the test tube and being isolated by filtra tion. The as-synthesised crystals contain nitrobenzene molecules in the void spaces. However, since nitrobenzene has a high relationship to the pores, target guests are poorly absorbed into the as-synthesised crystal. As such, a solvent exchange step that replaces nitrobenzene with an inert, noninteractive solvent is required prior to soaking the crystal in the target guest solution.Cyclohexane can be adopted as the inert solvent, while pentane withal proves useful for guest soaking at temperatures below 0oC. The solvent exchange step is carried out by soaking the crystal in the inert solvent for 7 days at 50oC. The success of the process can be monitered throughout by observing the disappearance of the signal at 1346 cm-1 in an Infrared (IR) spectrum, which can be assigned to nitrobenzene. Completion of the process is sustain by SC-XRD by the presence of ordered cyclohexane molecules in the pores. The sponge whitethorn now be used for guest absorption. This solvent exchange proc ess may complicate the refinement of the structure, since some nitrobenzene may reamin within the sponge structure after exchange (Vinogradova et al., 2014). This becomes an issue if the target guest molecule contains cyclohexyl or aromatic rings, as it may be difficult to distinguish the guest from residual solvent, especially if the site occupancy is low or the data quality is poor. go with with heavy use of crystallographic restraints, this increases the risk of misassignment of the desired guest molecule by using residual solvent electron density. Additionally, if the residual solvent and the guest interact similarly with the host, the likelihood of occupational disorder increases and making structure refinement much more challenging.Clardy and co-workers later reported a simpler and less timely preparation method for the synthesis of sponge 3 using similar conditions to those reported by Fujita and his team. (5sync) Instead of conducting the layer diffusion step with TTP in ni trobenzene, TTP is dissolved in chloroform. As such, the as-synthesised crystals of sponge 3 contain chloroform in the pores. Since chloroform has a much lower affinity for the solvent pores than nitrobenzene, the solvent exchange step can be omitted and the as-synthesised crystals used immediately. As well as saving 7 days of preparation by omitting the solvent exchange step, this method is also milder as it does not require the crystal to be heated for long periods of time. This reduces the chances of introducing imperfections in the crystal.This omission also minimises the number of solvents that the crystal is exposed to, reducing issues in structure refinement. Although some CHCl3 might remain within the sponge after guest inclusion, due to its longer C-Cl bond length (1.7) and larger Cl electron density, CHCl3 can still be observed. This greater electron density for CHCl3 exerts a larger influence on the structure factors relative to incorporated guest compared to nitrobenzene , however the benefits of CHCl3 usage override this issue.In addition to the desired crystals, this preparation method has been found to simultaneously form other crystalline structures. Firstly, a crystalline compound with the formula (ZnI2)3(TPT)2CHCl3n (2), having a much smaller pore size has been viewed. Fortunately, this crystalline structure can be easily distinguished from the desired structure from its syllable structure (Fig). A second undesired crystal has more recently been observed with consistently distinct unit parameter, but having indistinguishable morphology to the desired structure from its morphology (Fig). Both these crystals are believed to form due to slight changes in humidity and temperature as well as variations in mixing in the initial stages of the layering process. desired crystal. Both these crystals are believed to form due to slight changes in humidity and temperature as well as variations in mixing in the initial stages of the layering process.