D- and L-amino acids in synthetic peptides

The alpha carbon of an amino acid carries an amino group, a carboxyl group, a hydrogen and a side chain. When all four differ, the carbon is a stereocentre and the molecule exists as two non-superimposable mirror images, the L and D forms; this property is chirality. Glycine, whose side chain is a second hydrogen, is the one standard amino acid without it.

The D and L labels relate each amino acid to glyceraldehyde by the Fischer convention; they do not map one-to-one onto the R and S labels of the Cahn-Ingold-Prelog system. L-cysteine is R, because its CH2SH group outranks the carboxyl group (sulfur outranks oxygen), while every other chiral standard L-amino acid is S. Isoleucine and threonine have a second stereocentre in the side chain, so each has four stereoisomers.

D-amino acids by design

A residue is read as L unless it carries a D prefix, so D-Phe is D-phenylalanine. Four catalogue compounds are written with D-residues in their PubChem records; the sequences below are reference data for each compound, not measurements of any Solara lot.

PubChem gives no short form for PT-141 (CID 9941379); its row is transcribed from the record's systematic name, in which the ring is a lactam between Asp and Lys.

Aib is 2-methylalanine, 2-Nal 3-(2-naphthyl)alanine, Dmt 2,6-dimethyltyrosine and Nle norleucine. Aib has two methyl groups on its alpha carbon, so like glycine it is achiral: its mirror image is the same molecule.

Racemization during synthesis

Racemization converts one configuration at an alpha carbon into a mixture of both. Within a peptide, where every other stereocentre is unchanged, the product is a diastereomer of the target (the cited review calls this epimerization): the same formula and mass, but a different compound.

It happens mainly when a protected amino acid is activated for coupling: activation makes the alpha hydrogen acidic, and a base in the coupling mixture can remove it, directly or via a cyclic oxazolone. The planar intermediate takes a proton back from either face, so the residue couples as a mixture of L and D. Cysteine and histidine are among the residues most prone to it.

Acid hydrolysis for amino acid analysis, commonly 6 N HCl at 110 °C, can also invert residues, so methods correct for it.

How chiral purity is measured

Chiral purity is the share of each amino acid in its intended configuration. A single inverted residue gives a diastereomer with the same mass as the target, so intact mass spectrometry cannot see it. Reverse-phase HPLC sometimes separates diastereomers, but not reliably.

The dependable route is hydrolysis, then chiral analysis of the free amino acids. Marfey's reagent, which carries an L-alanine amide group, derivatizes the amino acids so that D and L forms become diastereomers, which an ordinary reverse-phase column separates; gas chromatography on a chiral column is one alternative. Hydrolysis in deuterated acid (DCl in D2O) tags any residue that inverts during hydrolysis with deuterium, which a mass detector can tell from a D-residue already present. Either route gives a D:L ratio per kind of amino acid, not the position of an inverted residue.