For decades, small molecule X-ray analysis has been the definitive tool for structural analysis. (9) This technique, however, is not without limitations. The process is considered by many an, where the production of high-quality crystals suitable for X-ray diffraction requires uncodified “tricks of the trade” as well as a certain amount of luck! Additionally, even after a substance has been successfully crystallized, there is no guarantee that the particular crystal form will be amenable to X-ray diffraction. Since crystal growth is both a slow and arduous process, X-ray diffraction has not been an effective tool for rapid, on-the-fly structural determination of small molecules. For this reason, X-ray diffraction is generally not implemented as a routine analytical tool for the practicing organic chemist, despite the fact that the structural data provided are far superior to any other characterization method to date.

The history of organic chemistry closely parallels the development of new methods for structural characterization. The earliest studies were driven by melting point determination, and over the past 175 years more complex methods for interrogation of structure have been developed. Techniques such as polarimetry, (1) UV–vis, (2) and infrared spectroscopy, (3) coupled with electron paramagnetic resonance, (4) vibrational circular dichroism, (5) circular dichroism, (6) and mass spectrometry (7) have been commonly employed over the years, dramatically expanding our ability to assign structures. In the past 50 years, however, the explosion of NMR spectroscopy (8) and the accompanying abundance of individual NMR experiments have produced a wealth of detailed structural information for organic chemists. Indeed, NMR is a mainstay in chemistry and the most predominant method employed in both routine synthetic chemistry experiments and in advanced structural elucidation of complex small molecules. In the current state of the art, only single crystal X-ray diffraction holds a higher place in terms of precision, producing unequivocal structural information about the position, orientation, connectivity, and placement of individual atoms and bonds within a given molecule.

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Herein, we employ the recently developed electron cryo-microscopy (CryoEM) method microcrystal electron diffraction (MicroED) (10) to address the long-standing need for fast and reliable structure determination in organic chemistry. Recently, electron diffraction was used to solve the structure of a methylene blue derivative, although no scope studies were undertaken to allow the reader to assess the applicability of the methodology. (11) Moreover, a specialized detector was used for their experiments, limiting the broad adaptability of their approach to the wider synthetic community. (11) We demonstrate that with minimal sample preparation and experiment time, simple powders and seemingly amorphous materials (in some cases, solids isolated via silica gel chromatography and rotary evaporation) could be directly used in MicroED studies, leading to rapid, high-quality structural elucidation of several classes of complex molecules with atomic resolution, in many cases better than 1 Å. Moreover, we utilize a commercially available microscope that is already in use at universities around the world. MicroED has the potential to dramatically accelerate and impact the fields of synthetic chemistry, natural product chemistry, drug discovery, and many others by delivering rapid, high-resolution atomic structures of complex, small molecules with minimal sample preparation or formal crystallization procedures.

1) as a model system (1 was resolved to an impressive 1 Å resolution. The entire process, from bottle to structure, was easily accomplished in less than 30 min. The applicability of MicroED was initially tested on the naturally occurring steroid progesterone () as a model system ( Figure 1 ). The sample was obtained as a powder from chemical supplier Preparations Laboratories Inc. (we estimate the bottle to be more than 20 years old). Small quantities of the seemingly amorphous solid were transferred directly from the bottle onto glass cover slides and ground between another slide to produce a fine powder. The powder was then deposited on a holey carbon–copper grid, cooled to liquid nitrogen temperatures, and transferred to a cryoelectron microscope operating at an acceleration voltage of 200 kV (Thermo Fisher Talos Arctica). An overview of the preparation is shown in Figure 1 . Upon imaging, thousands of nanocrystals were easily discernible on the grid surface providing ample nanocrystals to investigate for diffraction. Typically, for samples such as these, the vast majority of nanocrystals diffracted to ∼1 Å resolution or better ( Figure 1 ). Through continuous rotation of the sample stage, 140 degrees of diffraction data could be collected from a single nanocrystal, (12) while the improved autoloader and piezo stage of the Talos Arctica allowed us to travel through the zero degree point without introducing errors in crystal position. Typically, the stage was rotated at approximately 0.6 deg/s, and an entire data set was collected in less than 3 min as a movie ( Video 1 ) using a bottom mount CetaD CMOS detector (Thermo Fisher) fitted with a thick scintillator for diffraction studies. Software adapted from previous studies (13) was used to convert the diffraction movie frames into SMV format for expeditious processing in the readily available XDS software package commonly used for X-ray crystallography. (13) By collecting data from just a single nanocrystal, the structure of steroidwas resolved to an impressive 1 Å resolution. The entire process, from bottle to structure, was easily accomplished in less than 30 min.

Figure 1 Figure 1. Process of applying MicroED to small molecule structural analysis. Here commercial progesterone (1) was analyzed, and an atomic resolution structure was determined at 1 Å resolution. Grid holes are 1 μm in diameter.

2) and ibuprofen (3) in rapid succession. Just as impressively, structures of the sodium channel blocker carbamazepine (4) and the macrocyclic polypeptide antibiotic thiostrepton (5) were also determined from seemingly amorphous powders used as received from Sigma-Aldrich. We went on to further study several commercially available natural products and derivatives. Once again, compounds were used as received, without any crystallization, to yield atomic resolution structures of biotin (6), ethisterone (7), cinchonine (8), and brucine (9). Of the 11 different commercial bioactives examined, all 11 yielded processable MicroED data. Of those 11 compounds, 10 were amenable to rapid structure determination by direct methods, Encouraged by these results, we wanted to investigate a wide range of natural products to fully explore the scope and applicability of this powerful structural determination method for small molecules ( Figure 2 A). The Talos Arctica was particularly amenable to our studies as it is capable of storing up to 12 different grids at once, providing effortless swapping of sample grids for rapid investigation of multiple compounds. Once a reliable sample prep routine had been established, over-the-counter medications were purchased from local pharmacies for investigation. Tablets of CVS-branded acetaminophen and Kroger-branded ibuprofen were crushed using a mortar and pestle, and the ground powder was placed on electron microscopy grids as described above. Despite the heterogeneity of such pharmaceuticals, which typically include a multitude of coatings, binders, and other formulation agents, we were astonished to obtain such clearly resolved atomic resolution structures of both acetaminophen () and ibuprofen () in rapid succession. Just as impressively, structures of the sodium channel blocker carbamazepine () and the macrocyclic polypeptide antibiotic thiostrepton () were also determined from seemingly amorphous powders used as received from Sigma-Aldrich. We went on to further study several commercially available natural products and derivatives. Once again, compounds were used as received, without any crystallization, to yield atomic resolution structures of biotin (), ethisterone (), cinchonine (), and brucine (). Of the 11 different commercial bioactives examined, all 11 yielded processable MicroED data. Of those 11 compounds, 10 were amenable to rapid structure determination by direct methods, (14) while one was determined by molecular replacement. (15) As mentioned previously, all structures were obtained without any crystallization attempts or chemical modifications to compounds examined. While these pharmaceutical and commercial natural products were likely recrystallized for purification purposes by the manufacturer, the powders examined by MicroED possessed nanocrystals a billionth the size (∼100 nm) of crystals typically needed for X-ray crystallography. This was powder to structure.

Figure 2 Figure 2. Different types of small molecules solved by MicroED. (A) Several pharmaceuticals, vitamins, commercial natural products, and synthetic samples resolved through MicroED. (B) Example of an amorphous film utilized in this study leading to 1 Å resolution data. (C) Protons could be observed for several compounds through MicroED. Green density are F o – F c maps showing positive density belonging to hydrogen atoms of the molecule.

10 and 11, 10), an alkaloid natural product synthesized by our laboratories,10) and carbamazepine (4) after refinement showed protons associated with almost all atoms in these molecules ( Next we decided to investigate compounds that were never crystallized, but instead were purified by flash column chromatography. Since silica gel chromatography is the most common method of purification in early stage research for complex molecules in drug discovery, natural product isolation efforts, and synthesis efforts in general, we were interested to see whether solid samples prepared in such a way would be amenable to analysis by MicroED. Four compounds, purified by chromatographic methods, were collected from our laboratories, and samples of these seemingly amorphous solids were analyzed. Here, two of four compounds diffracted, yielding atomic resolution structures at or below 1 Å (and 2 Figure A,B). While the success rate for these compounds was 50%, it is worthy to note that no crystallization procedures were employed in the isolation of these materials. Notably, (+)-limaspermidine (), an alkaloid natural product synthesized by our laboratories, (16) was resolved from a residue of only milligram quantities of material following flash chromatography and rotary evaporation from a scintillation vial ( Figure 2 B). Furthermore, while it is extremely challenging to observe protons in X-ray structures, electrons interact with matter more strongly than X-rays and are affected by charge, making them relatively common to observe in MicroED data. (17−21) For all structures resolved from our samples, at least some, if not most, protons could be observed on the molecule. In particular, the density maps obtained for limaspermidine () and carbamazepine () after refinement showed protons associated with almost all atoms in these molecules ( Figure 2 C).

4, 6, 8 and 9, cf. Astounded by the ease with which such high-quality data were obtained and the apparent generality of MicroED to small molecules, we undertook studies to examine heterogeneous samples (mixtures of compounds). In the case of heterogeneous samples, single crystal X-ray diffraction precludes the study of mixtures, and NMR is poorly suited for this task. For this experiment, mixtures of four compounds (and, cf. Figure 3 ) were crushed together and deposited on a holey copper–carbon. Several crystal forms belonging to the different compounds in the mixture were visually identified on the grids ( Figure 3 ). MicroED data were collected from several nanocrystals, and the identity of each species was resolved within minutes by confirmation of unit cell parameters. After compound identification, atomic resolution structures could be rapidly determined for all small molecules present in the mixture ( Figure 3 ). (Note: no unexpected or unusually high safety hazards were encountered.)

Figure 3 Figure 3. Identification of compounds from heterogeneous mixtures. An EM grid was prepared as above with biotin, brucine, carbamazepine, and cinchonine powders mixed together. All four compounds identified by unit cell parameters using MicroED data from within the same grid square. All structures were solved to ∼1 Å resolution. Grid holes are 2 μm in diameter.