Stereoisomers: chirality, enantiomers and racemic mixtures

Stereoisomers are molecules with the same formula and the same bonds, arranged differently in space. Chirality is the property of a molecule that can't be laid over its own mirror image, like a left and a right hand; the two mirror-image forms are called enantiomers. They weigh exactly the same, yet they are different molecules. This guide explains chirality, the types of stereoisomer, the naming systems for them, and why a mirror-image impurity in a peptide is one of the hardest to find.

For the short version as it applies to amino acids, see the L- and D- section of the 20 amino acids. Terms are defined in the glossary.

What are stereoisomers?

Isomers are molecules with the same formula but a different arrangement of atoms. There are two broad kinds:

• Structural isomers have their atoms joined in a different order.
• Stereoisomers have their atoms joined in the same order, but arranged differently in space.

You can't turn one stereoisomer into another just by rotating the molecule or twisting it around a single bond. Getting from one to the other means breaking and remaking a bond.

Chirality and stereocentres

A chiral object can't be laid exactly over its own mirror image. Hands are the everyday example: a left glove won't fit a right hand. An object that can be laid over its mirror image, such as a plain mug, is achiral.

In molecules, chirality most often comes from a stereocentre: a carbon atom bonded to four different groups. Those four groups can be arranged in two ways that are mirror images of each other. If any two of the groups are the same, the two arrangements are identical and there is no stereocentre.

Every standard amino acid except glycine has a stereocentre at its alpha carbon. Glycine's side chain is a second hydrogen, so its alpha carbon has two identical groups.

On a skeletal formula, the arrangement at a stereocentre is shown with wedges and hashes. See reading a structure diagram.

Enantiomers (optical isomers)

Enantiomers are a pair of stereoisomers that are mirror images of each other. L-alanine and D-alanine are enantiomers.

Enantiomers have identical physical properties in an ordinary setting:

• the same mass and formula,
• the same melting point, boiling point and solubility,
• the same retention time on an ordinary HPLC column.

They differ in only two kinds of situation:

• With polarised light. Each one rotates plane-polarised light, by the same amount but in opposite directions. A compound that does this is called optically active, which is why enantiomers are also called optical isomers.
• With other chiral things. A chiral reagent, a chiral column or an enzyme can tell them apart, in the same way a right-handed glove tells your hands apart.

Diastereomers

Diastereomers are stereoisomers that aren't mirror images. They appear when a molecule has more than one stereocentre and some, but not all, are flipped.

A molecule with n stereocentres can have up to 2ⁿ stereoisomers:

Unlike enantiomers, diastereomers have different physical properties. Their melting points, solubilities and retention times can all differ, although sometimes only slightly.

This matters for peptides. If one residue in an all-L peptide is flipped to D, the result isn't the peptide's enantiomer. It is a diastereomer, because the other residues are still L. So an ordinary reversed-phase HPLC column can sometimes separate it from the main peptide, but not always.

Naming systems: L/D, R/S and (+)/(−)

Three different systems label stereoisomers, and they don't line up with each other.

Some points that often confuse:

• L doesn't mean (−). L and D describe the arrangement, not the direction of rotation. Some L-amino acids rotate light clockwise and some anticlockwise, and the direction can change with the solvent and pH.
• L doesn't always mean S. Most L-amino acids are S. L-cysteine is R, because its sulfur changes the priority order, not because its shape is different.
• D-amino acids are written with a prefix. A residue in a sequence with no prefix is assumed to be L. D-residues are written as, for example, D-Ala or with a lower-case one-letter code, as in the D-Ala2 substitution in CJC-1295. See amino acid codes.

Racemic mixtures

A racemic mixture, or racemate, is a 50:50 mix of two enantiomers. Their rotations cancel, so it has no overall effect on polarised light.

Ordinary chemical reactions that create a stereocentre from scratch, without any chiral influence, give a racemic mixture. The two arrangements are equally likely. That is why making a single enantiomer needs either a chiral starting material, a chiral catalyst, or a separation step.

Racemisation is the process of a pure enantiomer turning partly into its mirror image, heading towards a racemic mixture. Heat, strong acid or base, and some reaction conditions can all cause it.

Racemisation in peptide synthesis

Peptide synthesis starts from amino acids that are already a single enantiomer, almost always L. The risk is that a residue flips during the synthesis.

It happens mainly at the coupling step, when the carboxyl group of the incoming amino acid is activated so it will react. Strong activation can let the activated residue form a ring intermediate called an oxazolone. While in that form, its alpha carbon can lose and regain a hydrogen and flip to the D form (Molecules, 2023).

Some residues are more prone to this than others. Histidine, cysteine and serine are among those most often tested for it (Molecules, 2023). Chemists limit it by choosing coupling reagents and additives carefully, and by keeping conditions mild. See solid-phase peptide synthesis.

When a single residue in a peptide flips like this, the term is epimerisation. The product, called an epimer, differs at just one of several stereocentres.

Why mirror-image impurities are hard to detect

A peptide with one D-residue in place of an L-residue has:

• exactly the same mass, so mass spectrometry can't tell them apart,
• the same formula and sequence, so amino acid analysis by the standard method gives the same result,
• very similar properties, so on HPLC it may run just beside the main peak, or under it.

That makes it one of the few impurities that can hide in a purity figure. See reading impurity peaks for the "no mass change" impurities, and reading a chromatogram for how co-eluting peaks are missed.

Chiral methods

Finding a D-residue needs a method that is itself chiral:

• Chiral chromatography uses a column with a chiral stationary phase, so the two forms stick to it differently.
• Derivatisation reacts the sample with a chiral reagent first. Each enantiomer becomes a different diastereomer, which an ordinary column can separate. Marfey's reagent, introduced in 1984 to measure D-amino acids, works this way (Marfey, 1984).
• Chiral amino acid analysis breaks the peptide down into its amino acids and then measures the L:D ratio of each one with a chiral method.

These tests are specialised and aren't part of a standard purity and identity certificate.

Related guides

For L and D amino acids in brief, see the 20 amino acids. For how a structure is drawn, including wedges and hashes, see reading a structure diagram. For what an HPLC purity figure does and doesn't cover, see HPLC purity explained. An independent lab tests every lot we sell for purity by HPLC and identity by mass spectrometry; chiral purity isn't one of those tests. See Quality.