New solution for structural analysis of microvolume samples|MicroED

Structure elucidation is an important part of drug production and research, but in the early stages of drug discovery, large quantities of high-purity samples are often not available for single-crystal culture and structure elucidation. This slows down the overall drug discovery process to a certain extent, and scientists may have to spend a lot of effort on optimizing the process conditions to obtain a large number of high-purity samples to finally achieve the goal of structural analysis. Paclitaxel is a natural secondary metabolite isolated and purified from the bark of the redbud, and has been clinically proven to be effective against ovarian, uterine, and breast cancers, which have a high incidence of cancer. The paclitaxel required for clinical and scientific research is mainly extracted directly from redbud, but the content of paclitaxel in the plant is quite low (even the bark of short-leaved redbud, which is recognized to have the highest content, contains only 0.069%), and the extraction rate is less than 0.01%. Although single-crystal X-ray diffraction is the most common tool for the molecular resolution of drugs, single-crystal X-ray diffraction analysis requires high crystal size and quality, and the cultivation of single crystals suitable for single-crystal X-ray diffraction analysis requires sufficient samples and a lot of time to figure out the crystallization conditions (Figure 1). Therefore, for those drugs with a small number of samples, it is clear that single-crystal X-ray diffraction is no longer applicable.

Figure 1. Flow chart of single-crystal culture.

Figure 2 Workflow diagram of MicroED4.

The MicroED technique offers unparalleled advantages over single-crystal X-ray diffraction in the structural resolution of paclitaxel. Single-crystal X-ray diffraction requires the cultivation of single crystals of the right size, which requires sufficient samples to figure out the right crystallization conditions. Take the diffusion method as an example: a small bottle containing 1 mL of paclitaxel saturated solution is placed in a large bottle containing 20 mL of undesirable solvent, and the large bottle is sealed and left to stand. Depending on the types of good and bad solvents, a sample volume of at least about 1600 mg of paclitaxel was required for simultaneous single-crystal incubation experiments in 80 groups. After 1 week, tiny crystals were found by polarized light microscopy in several solvent systems, but its small size of about 200 nm made it difficult to satisfy the need for single-crystal X-ray diffraction. After 8 weeks, crystals capable of single-crystal X-ray diffraction were still not obtained.

Table 1. Sample information of some single-crystal cultures.

In this case, it is necessary to change to another solvent system and re-culture the single crystals, requiring a larger amount of paclitaxel samples. For the MicroED technique, since the nanometer-sized crystals are already able to meet the testing requirements, the need for paclitaxel samples is greatly reduced, and only 100 mg of sample is required to meet the crystal culture requirements. Meanwhile, for nanocrystals that cannot be tested by single-crystal X-ray, their diffraction data can be collected by MicroED technology, and then their crystal structures can be obtained by analytical reduction (Figure 3). In summary, the advantages of the MicroED technique are: (i) the sample requirement is small (typically one-tenth of the sample of a single crystal culture), (ii) the size requirement is relatively low, and nanocrystals can meet the testing requirements
Figure 3. Morphology and structure of paclitaxel.

MicroED technology requires only a small amount of sample (~100 mg) to culture nanocrystals and obtain high-quality diffraction data, which greatly reduces the need for sample quantity in the single crystal culture process and is very suitable for some synthetic drugs or natural products with small sample quantity, providing a new way for structural analysis of synthetic drugs and natural products.

ReadCrystal is a high-tech biopharmaceutical company based on structural analysis and AI drug screening technology, providing new drug discovery services, structural biology, and drug solid-state research services for drug discovery. With MicroED, ReadCrystal can achieve structural analysis of 100 nm crystals.

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