Alpha Amino Acids, with the exception of glycine, are chiral molecules that are well known for only being present in their L-forms, rather than their D-forms, in mammalian proteins and peptides. L- and D- amino acids are stereoisomers (with configurations based on comparison with glyceraldehyde defined as D) that differ in the location of the amino group on the alpha carbon, L-amino acids having it on the left side and D-amino acids on the right (see Figure 1).
D-forms of amino acids, although rare, have been shown to exist in nature and at least some play key biological roles such as D-serine in neurotransmission (1). However, it is the L-forms of the amino acids that are specifically bound to their tRNA carriers for the protein translation process. So, if this is the case then should we care about D-amino acids in proteins or peptides at all? The answer is, in fact, yes.
Firstly, whilst L-amino acids are nature’s “go-to” stereoisomer, D-amino acids have been shown to have biological significance in specific circumstances. As an example, D-amino acids may be specifically included in the sequence of chemically synthesized peptides in order to modify some aspect of their peptidic properties, such as increased resistance to proteolysis. This resistance to proteolysis has been demonstrated, for example, when D-amino acids were added to the termini of synthetic MUC2 epitope peptides in an antibody-binding investigation which also showed an increased half-life of the peptide in plasma (2).
Perhaps the best-known example of proteolytic resistance conferred by D-amino acid substitutions followed from the first discovery of natural Opioid Peptides in the brain, christened the Enkephalins, whose structures were determined by mass spectrometry in 1975 (3). It was quickly realized that they could not themselves be used as analgesics due to rapid proteolytic degradation. This triggered the development of a new field of Peptide Mimetics and the first in a long line of Peptide Analgesic compounds was the D-Ala-2 Met Enkephalin analogue DALA (4) which (by inhibition of proteolysis due to the presence of the D-Amino Acid) produced potent long-lasting analgesia in model tissue systems.
A more recent example of developments in the Opioid Peptide field is the drug Difelikefalin, that acts as an agonist of the k-opioid receptor and is used to treat itching in adults with chronic kidney disease; it is a chemically synthesized product that contains four D-amino acids.
So, if D-amino acids are to be specifically incorporated into peptides then a definitive method is needed to distinguish these from their L-forms in the final product and thus demonstrate successful synthesis, and that the structure of the product is as intended.
The simplest way to assess for D-amino acids is through amino acid analysis using a process that shows no chiral discrimination in terms of sample chemistry but that will allow D and L forms of the released amino acids to be separated and identified through on-line LC-MS using multiple reaction monitoring protocols.
This is a procedure that we have successfully implemented at BioPharmaSpec, allowing separation and identification of all L- and D- forms of the amino acids that we would see in the more typical amino acid analysis of only the L forms (for an example of D and L amino acid separation and identification see Figure 2). It should be noted that depending on strategy and conditions, some D-/L- conversion (racemization) occurs during the hydrolysis procedure so appropriate controls must be used within the experiment to identify the naturally present D-amino acid(s). Furthermore, and again depending on specific protocol particular amino acids may not be monitored, for example Glutamine may be converted to Glutamic acid, and likewise for Asparagine to Aspartic acid.
Of course, this analysis can be employed as part of a general structural assessment of a biopharmaceutical but has particular merit for those synthetic peptides where chirality of the amino acid needs to be confirmed. This D-/L- amino acid analysis could also form part of an impurity screening investigation of synthetic peptides to determine if any of the incorrect stereoisomer is present in the product.
Impurities arising from the synthesis process are not just associated with the actual product itself but can also be present in the product as a result of incomplete purification post-manufacture, so it is also important to consider an assessment of any residual chemicals that may be present from the synthesis procedure itself.
With increased interest in synthetically manufactured or modified peptides as therapeutic agents, the ability to monitor both L- and D-amino acid stereoisomers becomes essential.
Get in touch with us today to discuss how our expertise can support your amino acid analysis needs. Let’s ensure your product is exactly as intended.
