The fundamental concept of chirality

Chiral drugs refer to a pair of enantiomers that are mirror images of each other when a chiral center is introduced into the molecular structure. Each enantiomeric pair, when chemically pure, exhibits distinct physical and chemical properties (not just limited to optical activity) and can be named as R or S, D or L, P or M, or left-handed or right-handed, depending on different nomenclature conventions.

Currently, 50% of clinically used drugs are chiral drugs, and among the top ten globally sold drugs, 9 are chiral drugs. The prevalence of chiral drugs is due to the fact that the basic building blocks of drug targets, such as amino acids and nucleotides, are chiral structures.

Chirality is a crucial factor in molecular configuration. Organic compounds are categorized into various types of isomers in stereochemistry, including configurational isomers, geometric isomers, and optical isomers (enantiomers). The key to the formation of optical isomers lies in the molecule's asymmetry (chirality). Chiral molecules are organic compounds that cannot overlap with their mirror-image molecules.

Chiral molecules fall into three main categories:

Chiral molecules containing chiral atoms;

Axially chiral compounds (rotational isomers containing a chiral axis);

Optical isomers containing a chiral plane.

Common Naming Conventions for Chiral Molecules

R/S Notation:

The R/S system is a crucial nomenclature system for enantiomers. This method is based on the priority rules of atomic numbers. Each chiral center is assigned an R or S configuration based on the priority of substituents assigned by the system. When the orientation of the center places the lowest-priority substituent farthest from the observer, there are two possibilities: if the priorities of the remaining three substituents decrease in a clockwise direction, it is labeled as R (Rectus, Latin for right); if they decrease counterclockwise, it is labeled as S (Sinister, Latin for left). (R) or (S) is written in italics and enclosed in parentheses. If there are multiple chiral carbons, for example, (1R, 4S), the numbers specify the position of each configuration before the respective carbon.

Rules for determining R/S configuration:

a. Assign priorities to the groups connected to the chiral carbon in order.

b. Place the smallest group farthest from the observer.

c. Rotate the other three groups in a direction from largest to smallest. If the rotation is clockwise, the chiral carbon is in the R configuration; if counterclockwise, it is in the S configuration.

When comparing multi-atom groups, start by comparing the first atom. The atom with the higher atomic number comes first. If the first atoms are the same, compare the other atoms connected to the first atom. Start with the largest, then the middle, and finally the smallest until determining the order.

Figure 1: Absolute configuration of (S)-lactic acid

As shown in Figure 1, according to the rules of the R/S labeling method, the chiral center is connected to four groups: -H, -CH3, -OH, and -COOH. The first atoms connected to these groups are H, O, C, and C, respectively. Among them, the oxygen atom has the highest atomic number and is ranked first, while the hydrogen atom has the lowest atomic number and is ranked fourth. The remaining two are both carbon atoms, and their sizes cannot be compared directly. Therefore, we continue to compare down the chain. For the carbon in -CH3, the three atoms connected below, in descending order, are H, H, H. For the carbon in -COOH, the two atoms connected below, in descending order, are O, H. Starting with the largest atom, comparing -CH3's H with -COOH's O, O has a higher atomic number than H. Thus, -COOH has a higher priority than -CH3, and their priorities are 2 and 3, respectively.

Therefore, placing the smallest group farthest from the eyes, we can observe that the other three groups rotate in a counterclockwise direction, indicating an S configuration for the chiral carbon.

D/L Labeling Method:

Optical isomers can be named based on their spatial configuration of atoms. The D/L system (named after the Latin words dexter and laevus, meaning right and left) is implemented by associating the molecule with glyceraldehyde. Glyceraldehyde itself is chiral, and its two isomers are labeled as D and L. In this system, the naming of compounds is similar to glyceraldehyde, generating explicit names. The D/L label indicates the stereochemistry of a compound relative to the right- or left-handed enantiomer of glyceraldehyde. The right-handed isomer of glyceraldehyde is actually the D-isomer. Among the nineteen common L-amino acids in proteins, nine are right-handed (with a wavelength of 589 nm), and D-fructose, also known as levulose, is called a left-handed sugar because it is left-handed. An empirical rule for determining the D/L isomeric form of amino acids is the "CORN" rule.

Judgment rules for the D/L labeling method:

-COOH, -R, -NH2, and -H (where R is the side chain) are arranged around the chiral center carbon atom.

Hydrogen atoms are positioned away from the observer. If the groups CO → R → N are arranged counterclockwise around the central carbon atom, it is labeled as L-type. If arranged clockwise, it is labeled as D-type.

Figure 2: Absolute configuration of L-alanine

As shown in Figure 2, according to the D/L labeling method, the chiral center is connected to the -H, -COOH, -R, and -NH2 groups. Placing the smallest group (-H) farthest from the observer, we can see that the other three groups rotate around the carbon atom in the order of CO → R → N, and the rotation direction is counterclockwise, indicating an L configuration for the chiral carbon.

P/M Labeling Method:

For compounds containing a chiral axis, there is a lack of chiral centers corresponding to the use of the RS labeling method. We see that the chiral axis can be associated with a helix, and these molecules can be considered helical or helical propeller-like structures. The P/M labeling method is commonly used to label their configurations.

P/M Labeling Method Determination Rules:

Look from any end of the chiral axis and divide the compound into near and far groups along the chiral axis.

Determine the highest priority near group and the highest priority far group.

If moving from the highest priority near group to the corresponding far group requires a clockwise rotation, the helix is a right-handed helix and is described as P (or positive). Counterclockwise rotation means a left-handed helix and is specified as M (or negative).

Figure 3: P/M Labeling Method for Three Chiral Axis Compounds

As shown in Figure 3, according to the P/M labeling method, for compound A, the highest priority in the near group is the C atom, and in the far group, it is the Cl atom. By rotating in the C → Cl direction, the rotation is counterclockwise, indicating an M configuration. For compound B, the highest priority in the near group is the N atom, and in the far group, it is also the N atom. By rotating in the N → N direction, the rotation is clockwise, indicating a P configuration. For compound C, the highest priority in the near group is the C atom, and in the far group, it is also the C atom. By rotating in the C → C direction, the rotation is clockwise, indicating a P configuration.

Principles and Examples of Chiral Molecular Dynamics Refinement

Single crystal X-ray diffraction determines the absolute configuration of molecular structures through anomalous scattering. When the electrons on atoms are treated as free electrons, the scattering factor for X-rays is denoted as f0, and the phase angle difference between the scattering wave and the incident wave is a fixed value (π). However, different atoms exhibit varying abilities to bind electrons, leading to differences in the scattering capability of outer electrons compared to free electrons and resulting in a certain drift in the scattering phase angle. This phenomenon is wavelength-dependent and known as anomalous scattering.

On the other hand, MicroED (Micro Electron Diffraction) determines the absolute configuration of molecular structures through dynamic refinement. Compared to X-rays, electrons have a shorter wavelength and interact with matter many times stronger than X-rays, making them suitable for studying crystal materials at the nanometer level that X-ray diffraction cannot reach. Even in extremely small regions, strong electron diffraction can be obtained, making it particularly suitable for the analysis and identification of crystal phases in micro and nano regions. However, this advantage of electron diffraction also poses a challenge, as the diffraction patterns collected under TEM (Transmission Electron Microscopy) exhibit stronger dynamic effects, which can be a hindrance to structure resolution, as shown in Figure 4.

Figure 4 Significant Dynamic Effects Caused by Samples of Different Thicknesses

For chiral structures, there is a significant difference in the dynamic effects produced by the same compound in opposite chirality. When resolving the relative configuration of chiral compounds, we can use the differences in their dynamic effects to confirm the absolute configuration of chiral compounds through dynamic refinement.

Firstly, during data collection, we incorporate Precession Electron Diffraction (PED), which can average the impact of dynamic effects on diffraction patterns, as shown in Figure 5；

Figure 5 Schematic Diagram of the Working Principle of PED

1.Next, due to factors such as crystal thickness, mosaicity, bending, defects, etc., which are crucial for dynamic effects, during data processing, diffraction data suitable for dynamic refinement are selected based on the integration of diffraction point intensities in a double-hump diagram;

2.Finally, using the selected diffraction data, refine the compounds with different chiralities separately. The difference between the two makes it easy to determine the absolute configuration of unknown chiral compounds. Figure six illustrates 75 successful cases where the absolute configuration was determined through dynamic refinement.

Figure 6: Absolute Configuration Refinement Cases