Despite the dominance of L-amino acids, D-amino acids are not mere curiosities. They have been found in various biological systems, often playing critical roles.
D-Amino Acids in Nature
Previously referred to as “unnatural” amino acids, D-amino acids are not entirely accurate. Although L-configuration amino acids dominate the proteins and peptides in nature, organisms can still produce bioactive peptides and proteins containing D-amino acids. These peptides and proteins are synthesized either through the direct coupling of free D-amino acids mediated by non-ribosomal peptide synthetases (NRPS) or through D-modification of L-polypeptide precursors – also known as post-translational L-amino acid epimerization. The former mechanism is unique to prokaryotes, while the latter occurs in both prokaryotic and eukaryotic cells.
One of the best examples of D-amino acids found in natural biological structures is the peptidoglycan of microorganisms. D-alanine and D-glutamic acid are essential components of the cell walls of eubacteria, helping them resist hydrolysis by peptidases. D-amino acids are also found in yeast cells, where they help spores resist proteolytic enzymes. Although natural D-amino acids are rarely found in receptor proteins, structural proteins, and immune proteins, they can still be found in peptide antibiotics, hormones, neuropeptides, hepatotoxins, and opioid peptides.
Scientists have discovered peptide antibiotics containing D-amino acids with antimicrobial properties in many microorganisms. The structure and synthesis of peptide antibiotics have been well-studied in some members of the genus Bacillus. The therapeutic effects of cyclic antibiotics such as gramicidin S and polymyxin B are strongly correlated with the D-phenylalanine contained in their structures. The synthesis of all these peptides is non-ribosomal. Their antibiotic mechanisms generally rely on the formation of transmembrane channels or the inhibition of cell wall synthesis.
In eukaryotic cells, scientists have also discovered bioactive peptides containing D-amino acids, which are formed by post-translational modification of precursors composed of L-amino acids. D-amino acid peptides with opioid-like or antimicrobial activity have been isolated from the skin secretions of invertebrates and amphibians. Conotoxins, venomous peptides from predatory marine gastropods of the family Conidae, contain D-tryptophan or D-leucine and can cause fish death and even have a fatal effect on mammals by blocking neuromuscular transmission. The presence of D-amino acids has also been detected in peptide hormones of some arthropods, such as the venom of the North American funnel-web spider (Agelenopsis aperta), which contains multiple protein toxins that can block calcium channels.
Extensive studies on mammals and birds have shown that free D-amino acids exist in the tissues and biofluids of vertebrates, with D-aspartic acid and D-serine having the highest concentrations. D-aspartic acid is widely distributed throughout the central nervous system and in the endocrine peripheral organs of rats, humans, and chicken embryos. The localization of D-aspartic acid and D-serine in specific regions of the brain suggests that these D-amino acids may act as neurotransmitters or neuromodulators.
Due to the frequent detection of D-amino acids in natural antibiotics isolated from microorganisms, invertebrates, and amphibians, they are often used as the basis for the development of antimicrobial drugs. Among the antibiotics based on L/D-amino acids, well-known examples include tyrothricin (Bacillus brevis), D-cycloserine (Streptomyces orchidaceus), daptomycin (Streptomyces roseosporus), benzylpenicillin (Penicillium chrysogenum), actinomycin D (Streptomyces parvullus), and colistin (Streptomyces capreolus). These antibiotics can be classified into two categories based on whether they are primarily composed of protein amino acids or non-protein amino acids. For example, polymyxin and actinomycin D are mainly composed of protein amino acids, with only a few amino acids having the D configuration. Such antibiotics can have linear and cyclic structures. In most cases, antibiotics primarily composed of non-protein amino acids have a complete cyclic structure. At the same time, their molecular mechanisms of action are very diverse, ranging from dephosphorylation of bacterial membrane phospholipids (bacitracin A) to the formation of pseudo-ion channels that disrupt ion transmembrane transport (tyrothricin). The long-term cytotoxicity of these peptide antibiotics may be related to isomeric toxicity and the resistance of D-amino acid peptide bonds to hydrolysis.
All-D-type peptide enantiomers can exhibit properties different from their L-type counterparts. All-D theta-defensins, these cationic peptides are more stable than their L-type enantiomers and exhibit antiviral activity in vitro. Amino acid racemization can occur with the aging of organisms, which can occur in proteins such as osteocalcin, collagen, myelin, and β-amyloid. For example, the formation of cataracts may be related to the racemization of aspartic acid. In addition, amino acid racemization has been linked to Alzheimer’s disease, chronic kidney disease, and certain cancers.
D-Amino Acids in Peptide Drugs The introduction of D-amino acids into peptide drug molecules can effectively alleviate the enzymatic degradation of drug molecules by peptide hydrolases, thereby prolonging the half-life of drug molecules. D-proteins have a Ramachandran space opposite to that of L-proteins, so they can avoid many (unfavorable) interactions with natural proteins in terms of conformation. For example, D-proteins and peptides are highly resistant to degradation by normal cellular mechanisms. This is because the active sites of proteases are highly specific, and D-peptides cannot be broken down by these hydrolases due to their spatial structure and incompatibility with the corresponding hydrolases. In the development of peptide drugs, L-peptides are easily degraded by cells and usually have poor bioavailability. Theoretically, D-configuration peptides may have better pharmacological effects. If D-type peptides can interact with disease-related target receptors, coupled with their biological stability, they will be a very attractive candidate drug.
The multifunctionality and stability of D-type peptides have opened up possibilities for drug development, and many D-type peptide drugs are currently under investigation for the treatment of Alzheimer’s disease, AIDS, malaria, and other diseases. One of the earliest therapeutic applications of D-type peptides was to inhibit HIV entry into cells. Researchers have designed many D-type peptides to mimic the HIV-1 binding pocket and have achieved great success in inhibiting HIV infection. Some synthetic D-type peptides can also bind to Alzheimer’s amyloid-β peptide (Aβ), in which case the D-type peptide does not inhibit any receptors but stains the Aβ peptide and visualizes it, allowing doctors to see where the Aβ peptide accumulates in the brain.
Among the peptide drugs approved by the FDA in 2021, the appearance of D-amino acids is even more numerous. Among the peptide drugs approved by the FDA in 2021, voclosporin (D-Ala) and odevixibat (D-4-hydroxyphenylalanine) contain D-amino acids. While defelikefalin even adopts a full D-amino acid structure (D-Phe, D-Leu, D-Lys). Difelikefalin, marketed under the brand name Korsuva, is an analgesic opioid peptide used to treat moderate to severe pruritus.
Summary While nature has selected L-amino acids for the construction of life, D-amino acids also play significant roles in biological systems. They contribute to the stability and activity of many natural peptides and have great potential in drug design. With the increasing understanding of the structure and biological functions of D-amino acids in proteins and peptides, they will exert inreasing power in the therapeutical area.